U.S. patent application number 09/935384 was filed with the patent office on 2003-09-04 for nucleic acid and corresponding protein named 158p1h4 useful in the treatment and detection of bladder and other cancers.
Invention is credited to Afar, Daniel E. H., Challita-Eid, Pia M., Faris, Mary, Ge, Wangmao, Hubert, Rene S., Jakobovits, Aya, Levin, Elana, Raitano, Arthur B..
Application Number | 20030166526 09/935384 |
Document ID | / |
Family ID | 26921162 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166526 |
Kind Code |
A1 |
Challita-Eid, Pia M. ; et
al. |
September 4, 2003 |
Nucleic acid and corresponding protein named 158P1H4 useful in the
treatment and detection of bladder and other cancers
Abstract
A novel gene (designated 158P1H4) and its encoded protein are
described. While 158P1H4 exhibits tissue specific expression in
normal adult tissue, it is aberrantly expressed in multiple cancers
including set forth in Table 1. Consequently, 158P1H4 provides a
diagnostic and/or therapeutic target for cancers,. The 158P1H4 gene
or fragment thereof, or its encoded protein or a fragment thereof,
can be used to elicit an immune response.
Inventors: |
Challita-Eid, Pia M.;
(Encino, CA) ; Hubert, Rene S.; (Los Angeles,
CA) ; Raitano, Arthur B.; (Los Angeles, CA) ;
Afar, Daniel E. H.; (Brisbane, CA) ; Levin,
Elana; (Los Angeles, CA) ; Faris, Mary; (Los
Angeles, CA) ; Ge, Wangmao; (Culver City, CA)
; Jakobovits, Aya; (Beverly Hills, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
26921162 |
Appl. No.: |
09/935384 |
Filed: |
August 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60227098 |
Aug 22, 2000 |
|
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60282739 |
Apr 10, 2001 |
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Current U.S.
Class: |
435/6.14 ;
424/146.1; 514/19.3; 514/44A |
Current CPC
Class: |
C07K 14/47 20130101;
A61P 35/00 20180101 |
Class at
Publication: |
514/12 ; 514/44;
435/6; 424/146.1 |
International
Class: |
A61K 048/00; C12Q
001/68; A61K 039/395 |
Claims
1. A method for monitoring 158P1H4 gene products in a biological
sample from a patient who has or who is suspected of having cancer,
the method comprising: determining the status of 158P1H4 gene
products expressed by cells in a tissue sample from an individual;
comparing the status so determined to the status of 158P1H4 gene
products in a corresponding normal sample; and, identifying the
presence of aberrant 158P1H4 gene products in the sample relative
to the normal sample.
2. A method of monitoring the presence of cancer in an individual
comprising: performing the method of claim 1 whereby the presence
of elevated 158P1H4 MRNA or protein expression in the test sample
relative to the normal tissue sample provides an indication of the
presence or status of a cancer.
3. The method of claim 2, wherein the cancer occurs in a tissue set
forth in Table I.
4. A composition comprising: a substance that modulates the status
of 158P1H4 or a molecule that is modulated by 158P1H4 and thereby
modulates the status of a cell that expresses 158P1H4.
5. The composition of claim 4, further comprising a
pharmaceutically acceptable carrier.
6. A pharmaceutical composition that comprises the composition of
claim 4 in a human unit dose form.
7. A composition of claim 4 that comprises a 158P1H4-related
protein.
8. A composition of claim 4 that comprises an antibody or fragment
thereof that specifically binds to a 158P1H4-related protein.
9. A composition of claim 4 that comprises a polynucleotide that
encodes a single chain monoclonal antibody that immunospecifically
binds to an 158P1H4-related protein.
10. A composition of claim 4 that comprises a polynucleotide
comprising a 158P1H4-related protein coding sequence.
11. A composition of claim 4 that comprises an antisense
polynucleotide complementary to a polynucleotide having a 158P1H4
coding sequence.
12. A pharmaceutical composition of claim 4 that comprises a
ribozyme capable of cleaving a polynucleotide having 158P1H4 coding
sequence and a physiologically acceptable carrier.
13. A method of inhibiting growth of cancer cells that expresses
158P1H4, the method comprising: administering to the cells the
composition of claim 4.
14. A method of claim 13 of inhibiting growth of cancer cells that
express 158P1H4, the method comprising steps of: administering to
said cells an antibody or fragment thereof that specifically binds
to a 158P1H4-related protein.
15. A method of treating a patient with a cancer that expresses
158P1H4, the method comprising steps of: administering to said
patient a vector that comprises the composition of claim 9, such
that the vector delivers the single chain monoclonal antibody
coding sequence to the cancer cells and the encoded single chain
antibody is expressed intracellularly therein.
16. A method of claim 13 of inhibiting growth of cancer cells that
express 158P1H4, the method comprising steps of: administering to
said cells a polynucleotide comprising a 158P1H4-related protein
coding sequence.
17. A method of claim 13 of inhibiting growth of cancer cells that
express 158P1H4, the method comprising steps of: administering to
said cells an antisense polynucleotide complementary to a
polynucleotide having a 158P1H4 coding sequence.
18. A method of treating a patient with a cancer that expresses
158P1H4, the method comprising steps of: identifying that the
patient has a cancer the cells of which express 158P1H4;
administering to the patient a pharmaceutical composition of claim
12 that comprises a ribozyme capable of cleaving a polynucleotide
having a 158P1H4 coding sequence.
19. A method of generating a mammalian immune response directed to
158P1H4, the method comprising: exposing cells of the mammal's
immune system to an immunogenic portion of an 158P1H4-related
protein or a nucleotide sequence that encodes said protein, whereby
an immune response is generated to 158P1H4.
20. A method of delivering a cytotoxic agent to a cell that
expresses 158P1H4, said method comprising: providing a cytotoxic
agent conjugated to an antibody or fragment thereof that
specifically binds to 158P1H4; and, exposing the cell to the
antibody-agent conjugate.
21. A method of inducing an immune response to a 158P1H4 protein,
said method comprising: providing a 158P1H4-related protein that
comprises at least one T cell or at least one B cell epitope;
contacting the epitope with an immune system T cell or B cell
respectively, whereby the immune system T cell or B cell is
induced.
22. The method of claim 21, wherein the immune system cell is a B
cell, whereby the induced B cell generates antibodies that
specifically bind to the 158P1H4-related protein.
23. The method of claim 21, wherein the immune system cell is a T
cell that is a cytotoxic T cell (CTL), whereby the activated CTL
kills an autologous cell that expresses the 158P1H4 protein.
24. The method of claim 21, wherein the immune system cell is a T
cell that is a helper T cell (HTL), whereby the activated HTL
secretes cytokines that facilitate the cytotoxic activity of a CTL
or the antibody producing activity of a B cell.
25. An antibody or fragment thereof that specifically binds to a
158P1H4-related protein.
26. The antibody or fragment thereof of claim 25, which is
monoclonal.
27. A recombinant protein comprising the antigen-binding region of
a monoclonal antibody of claim 26.
28. The antibody or fragment thereof of claim 25, which is labeled
with a detectable marker.
29. The recombinant protein of claim 27, which is labeled with a
detectable marker.
30. The antibody fragment of claim 25, which is an Fab, F(ab')2, Fv
or sFv fragment.
31. The antibody of claim 25, which is a human antibody.
32. The recombinant protein of claim 27, which comprises murine
antigen binding region residues and human constant region
residues.
33. A non-human transgenic animal that produces an antibody of
claim 25.
34. A hybridoma that produces an antibody of claim 26.
35. A single chain monoclonal antibody that comprises the variable
domains of the heavy and light chains of a monoclonal antibody of
claim 26.
36. A vector comprising a polynucleotide that encodes a single
chain monoclonal antibody of claim 35 that immunospecifically binds
to a 158P1H4-related protein.
37. An assay for detecting the presence of a 158P1H4-related
protein or polynucleotide in a biological sample from a patient who
has or who is suspected of having cancer, comprising steps of:
contacting the sample with an antibody or another polynucleotide,
respectively, that specifically binds to the 158P1H4-related
protein or polynucleotide, respectively; and, determining that
there is a complex of the antibody and 158P1H4-related protein or
the another polynucleotide and 158P1H4-related polynucleotide.
38. The assay in accordance with claim 37 for detecting the
presence of a 158P1H4-related protein or polynucleotide in a
biological sample from a patient who has or who is suspected of
having cancer, comprising the steps of: obtaining a sample from a
patient who has or who is suspected of having cancer.
39. The assay of claim 37 for detecting the presence of an 158P1H4
polynucleotide in a biological sample, comprising: contacting the
sample with a polynucleotide probe that specifically hybridizes to
a polynucleotide encoding an 158P1H4-related protein having the
amino acid sequence SEQ ID NO.: 703; and, detecting the presence of
a hybridization complex formed by the hybridization of the probe
with 158P1H4 polynucleotide in the sample, wherein the presence of
the hybridization complex indicates the presence of 158P1H4
polynucleotide within the sample.
40. An assay for detecting the presence of 158P1H4 mRNA in a
biological sample from a patient who has or who is suspected of
having cancer, said method comprising: (a) producing cDNA from the
sample by reverse transcription using at least one primer; (b)
amplifying the cDNA so produced using 158P1H4 polynucleotides as
sense and antisense primers, wherein the 158P1H4 polynucleotides
used as the sense and antisense primers are capable of amplifying
the 158P1H4 cDNA contained within the plasmid as deposited with
American Type Culture Collection as Accession No. PTA-3136; and (c)
detecting the presence of the amplified 158P1H4 cDNA.
41. A composition comprising a polynucleotide from position number
4 through number 1386 of SEQ ID NO.: 702.
42. The composition of claim 41, wherein T is substituted with
U.
43. A composition comprising SEQ ID NO.: 702.
44. The composition of claim 43, wherein T is substituted with
U.
45. A composition comprising a polynucleotide that encodes an
158P1H4-related protein that is at least 90% homologous to the
entire amino acid sequence shown in SEQ ID NO.: 703.
46. An analog peptide of eight, nine ten or eleven contiguous amino
acids of SEQ ID NO.: 703
47. A polynucleotide that encodes an analog peptide of claim
46.
48. The composition of claim 45, wherein the polynucleotide encodes
an 158P1H4-related protein that is at least 90% identical to the
entire amino acid sequence shown in SEQ ID NO: 703.
49. A composition comprising a polynucleotide that encodes at least
one peptide set forth in Tables V-XVIII.
50. A composition comprising a polynucleotide that encodes a
peptide region of at least 5 amino acids of SEQ ID NO.: 703 in any
whole number increment up to 440 that includes an amino acid
position selected from: an amino acid position having a value
greater than 0.5 in the Hydrophilicity profile of FIG. 9, an amino
acid position having a value less than 0.5 in the Hydropathicity
profile of FIG. 10; an amino acid position having a value greater
than 0.5 in the Percent Accessible Residues profile of FIG. 11; an
amino acid position having a value greater than 0.5 in the Average
Flexibility profile on FIG. 12; or an amino acid position having a
value greater than 0.5 in the Beta-turn profile of FIG. 13.
51. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 41.
52. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 42.
53. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 43.
54. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 44.
55. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 45.
56. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 48.
57. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 49.
58. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 48.
59. A composition comprising a polynucleotide that encodes a
158P1H4-re late d protein whose sequence is encoded by the cDNAs
contained in the plasmid designated p158P1H4-EBB10 deposited with
American Type Culture Collection as Accession No. PTA-3136.
60. A composition comprising a polypeptide at least 90% homologous
to SEQ ID NO.: 703.
61. The composition of claim 60, wherein the polypeptide is at
least 90% identical to SEQ ID NO.: 703.
62. The composition of claim 61, wherein the polypeptide comprises
SEQ ID NO.: 703.
63. A composition comprising a CTL polypeptide epitope from SEQ ID
NO.: 703.
64. The composition of claim 63, wherein the CTL epitope comprises
a polypeptide selected from Tables V-XVIII.
65. A composition comprising a peptide region of at least 5 amino
acids of SEQ ID NO.: 703 in any whole number increment up to 440
that includes an amino acid position selected from: an amino acid
position having a value greater than 0.5 in the Hydrophilicity
profile of FIG. 9, an amino acid position having a value less than
0.5 in the Hydropathicity profile of FIG. 10; an amino acid
position having a value greater than 0.5 in the Percent Accessible
Residues profile of FIG. 11; an amino acid position having a value
greater than 0.5 in the Average Flexibility profile on FIG. 12; or
an amino acid position having a value greater than 0.5 in the
Beta-turn profile of FIG. 13.
66. A composition comprising a 158P1H4-related protein whose
sequence is encoded by the cDNAs contained in the plasmid
designated p158P1H4-EBB10 deposited with American Type Culture
Collection as Accession No. PTA-3136.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Applications 60/227,098, filed Aug. 22, 2000, and No.
60/282,739, filed Apr. 10, 2001, the entire contents of which are
incorporated herein by reference as if fully set forth.
FIELD OF THE INVENTION
[0002] The invention described herein relates to a novel nucleic
acid sequence and its encoded protein, referred to as 158P1H4, and
to diagnostic and therapeutic methods and compositions useful in
the management of various cancers that express 158P1H4. The
invention also relates to a novel nucleic acid sequence
158P1F4.
BACKGROUND OF THE INVENTION
[0003] Cancer is the second leading cause of human death next to
coronary disease. Worldwide, millions of people die from cancer
every year. In the United States alone, as reported by the American
Cancer Society, cancer causes the death of well over a half-million
people annually, with over 1.2 million new cases diagnosed per
year. While deaths from heart disease have been declining
significantly, those resulting from cancer generally are on the
rise. In the early part of the next century, cancer is predicted to
become the leading cause of death.
[0004] Of all new cases of cancer in the United States, bladder
cancer represents approximately 5 percent in men (fifth most common
neoplasm) and 3 percent in women (eighth most common neoplasm). The
incidence is increasing slowly, concurrent with an increasing older
population. In 1998, there was an estimated 54,500 cases, including
39,500 in men and 15,000 in women. The age-adjusted incidence in
the United States is 32 per 100,000 for men and 8 per 100,000 in
women. The historic male/female ratio of 3:1 may be decreasing
related to smoking patterns in women. There were an estimated
11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900
in women). Bladder cancer incidence and mortality strongly increase
with age and will be an increasing problem as the population
becomes more elderly.
[0005] Bladder cancers comprise a heterogeneous group of diseases.
The main determinants of disease control and survival are histology
and extent of disease. The main codes for these factors include
pathology classification, the International Classification of
Diseases-Oncology (ICDO), and staging classification of extent of
disease, the TNM classification.(Table XXI). For a general
discussion of bladder and other urogenital cancers, see, e.g.,
Volgelzang, et al, Eds. Comprehensive Textbook of Genitourinary
Oncology, (Williams & Wilkins, Baltimore 1996), in particular
pages 295-556.
[0006] Three primary types of tumors have been reported in the
bladder. The most common type of bladder cancer is Transitional
cell carcinoma (TCC); this accounts for about 90% of all bladder
cancers. The second form of bladder cancer is squamous cell
carcinoma, which accounts for about 8% of all bladder cancers where
schistosomiasis is not endemic, and approximately 75% of bladder
carcinomas where schistosomiasis is endemic. Squamous cell
carcinomas tend to invade deeper layers of the bladder. The third
type of bladder cancer is adenocarcinoma, which account for 1%-2%
of bladder cancers; these are primarily invasive forms of
cancer.
[0007] Bladder cancer is commonly detected and diagnosed using
cytoscopy and urine cytology. However these methods demonstrate
poor sensitivity. Relatively more reliable methods of detection
currently used in the clinic include the bladder tumor antigen
(BTA) stat test, NMP22 protein assay, telomerase expression and
hyaluronic acid and hyaluronidase (HA-HAase) urine test. The
advantage of using such markers in the diagnosis of bladder cancer
is their relative high sensitivity in earlier tumor stages compared
to standard cytology.
[0008] For example, the BTA stat test has 60-8-% sensitivity and
50-70% specificity for bladder cancer, while the HA-HAase urine
test shows 90-92% sensitivity and 80-84% specificity for bladder
cancer (J Urol 2001 165:1067). In general, sensitivity for stage Ta
tumors was 81% for nuclear matrix protein (NMP22), 70% for
telomerase, 32% for bladder tumor antigen (BTA) and 26% for
cytology (J Urol 2001 166:470; J Urol 1999, 161:810). Although the
telomeric repeat assay which measures telomerase activity is
relatively sensitive, instability of telomerase in urine presently
renders this detection method unreliable.
[0009] Most bladder cancers recur in the bladder. Generally,
bladder cancer is managed with a combination of transurethral
resection of the bladder (TUR) and intravesical chemotherapy or
immunotherapy. The multifocal and recurrent nature of bladder
cancer points out the limitations of TUR. Most muscle-invasive
cancers are not cured by TUR alone. Radical cystectomy and urinary
diversion is the most effective means to eliminate the cancer but
carry an undeniable impact on urinary and sexual function.
[0010] Intravesical bacilli Calmette-Guerin (BCG) is a common and
efficacious immunotherapeutic agent used in the treatment of
bladder cancer. BCG is also used as a prophylactic agent to prevent
recurrence of bladder cancer. However, 30% of patients fail to
respond to BCG therapy and go on to develop invasive and metastatic
disease (Catalona et al. J Urol 1987, 137:220-224). BCG-related
side effects have been frequently observed such as drug-induced
cystitis, risk of bacterial infection, and hematuria, amongst
others. Other alternative immunotherapies have been used for the
treatment of bladder cancer, such as KLH (Flamm et al. Urologe
1994; 33:138-143) interferons (Bazarbashi et al. J Surg Oncol.
2000; 74:181-4), and MAGE-3 peptide loaded dendritic cells
(Nishiyama et al. Clin Cancer Res 2001; 7:23-31). All these
approaches are still experimental (Zlotta et al. Eur Urol 2000;37
Suppl 3:10-15). There continues to be a significant need for
diagnostic and treatment modalities that are beneficial for bladder
cancer patients. Furthermore, from a worldwide standpoint, several
cancers stand out as the leading killers. In particular, carcinomas
of the lung, prostate, breast, colon, pancreas, and ovary are
primary causes of cancer death. These and virtually all other
carcinomas share a common lethal feature. With very few exceptions,
metastatic disease from a carcinoma is fatal. Moreover, even for
those cancer patients who initially survive their primary cancers,
their lives are dramatically altered. Many cancer patients
experience strong anxieties driven by the awareness of the
potential for recurrence or treatment failure. Many cancer patients
experience physical debilitations following treatment. Furthermore,
many cancer patients experience a recurrence.
[0011] Prostate cancer is the fourth most prevalent cancer in men
worldwide. In North America and Northern Europe, it is by far the
most common cancer in males and is the second leading cause of
cancer death in men. In the United States alone, well over 30,000
men die annually of this disease, second only to lung cancer.
Despite the magnitude of these figures, there is still no effective
treatment for metastatic prostate cancer. Surgical prostatectomy,
radiation therapy, hormone ablation therapy, surgical castration
and chemotherapy continue to be the main treatment modalities.
Unfortunately, these treatments are ineffective for many and are
often associated with undesirable consequences.
[0012] On the diagnostic front, the lack of a prostate tumor marker
that can accurately detect early-stage, localized tumors remains a
significant limitation in the diagnosis and management of this
disease. Although the serum prostate specific antigen (PSA) assay
has been a very useful tool, however its specificity and general
utility is widely regarded as lacking in several important
respects. While previously identified markers such as PSA, PSM,
PCTA and PSCA have facilitated efforts to diagnose and treat
prostate cancer, there is need for the identification of additional
markers and therapeutic targets for prostate and related cancers in
order to further improve diagnosis and therapy.
[0013] Renal cell carcinoma (RCC) accounts for approximately 3
percent of adult malignancies. Once adenomas reach a diameter of 2
to 3 cm, malignant potential exists. In the adult, the two
principal malignant renal tumors are renal cell adenocarcinoma and
transitional cell carcinoma of the renal pelvis or ureter. The
incidence of renal cell adenocarcinoma is estimated at more than
29,000 cases in the United States, and more than 11,600 patients
died of this disease in 1998. Transitional cell carcinoma is less
frequent, with an incidence of approximately 500 cases per year in
the United States.
[0014] Surgery has been the primary therapy for renal cell
adenocarcinoma for many decades. Until recently, metastatic disease
has been refractory to any systemic therapy. With recent
developments in systemic therapies, particularly immunotherapies,
metastatic renal cell carcinoma may be approached aggressively in
appropriate patients with a possibility of durable responses.
Nevertheless, there is a remaining need for effective therapies for
these patients.
[0015] An estimated 130,200 cases of colorectal cancer occurred in
2000 in the United States, including 93,800 cases of colon cancer
and 36,400 of rectal cancer. Colorectal cancers are the third most
common cancers in men and women. Incidence rates declined
significantly during 1992-1996 (-2.1% per year). Research suggests
that these declines have been due to increased screening and polyp
removal, preventing progression of polyps to invasive cancers.
There were an estimated 56,300 deaths (47,700 from colon cancer,
8,600 from rectal cancer) in 2000, accounting for about 11% of all
U.S. cancer deaths.
[0016] At present, surgery is the most common form of therapy for
colorectal cancer, and for cancers that have not spread, it is
frequently curative. Chemotherapy, or chemotherapy plus radiation
is given before or after surgery to most patients whose cancer has
deeply perforated the bowel wall or has spread to the lymph nodes.
A permanent colostomy (creation of an abdominal opening for
elimination of body wastes) is occasionally needed for colon cancer
and is infrequently required for rectal cancer. There continues to
be a need for effective diagnostic and treatment modalities for
colorectal cancer.
[0017] There were an estimated 164,100 new cases of lung and
bronchial cancer in 2000, accounting for 14% of all U.S. cancer
diagnoses. The incidence rate of lung and bronchial cancer is
declining significantly in men, from a high of 86.5 per 100,000 in
1984 to 70.0 in 1996. In the 1990s, the rate of increase among
women began to slow. In 1996, the incidence rate in women was 42.3
per 100,000.
[0018] Lung and bronchial cancer caused an estimated 156,900 deaths
in 2000, accounting for 28% of all cancer deaths. During 1992-1996,
mortality from lung cancer declined significantly among men (-1.7%
per year) while rates for women were still significantly increasing
(0.9% per year). Since 1987, more women have died each year of lung
cancer than breast cancer, which, for over 40 years, was the major
cause of cancer death in women. Decreasing lung cancer incidence
and mortality rates most likely resulted from decreased smoking
rates over the previous 30 years; however, decreasing smoking
patterns among women lag behind those of men. Of concern, although
the declines in adult tobacco use have slowed, tobacco use in youth
is increasing again.
[0019] Treatment options for lung and bronchial cancer are
determined by the type and stage of the cancer and include surgery,
radiation therapy, and chemotherapy. For many localized cancers,
surgery is usually the treatment of choice. Because the disease has
usually spread by the time it is discovered, radiation therapy and
chemotherapy are often needed in combination with surgery.
Chemotherapy alone or combined with radiation is the treatment of
choice for small cell lung cancer; on this regimen, a large
percentage of patients experience remission, which in some cases is
long lasting. There is however, an ongoing need for effective
treatment and diagnostic approaches for lunch and bronchial
cancers.
[0020] An estimated 182,800 new invasive cases of breast cancer
were expected to have occured among women in the United States
during 2000. Additionally, about 1,400 new cases of breast cancer
were expected to be diagnosed in men in 2000. After increasing
about 4% per year in the 1980s, breast cancer incidence rates in
women have leveled off in the 1990s to about 110.6 cases per
100,000.
[0021] In the U.S. alone, there were an estimated 41,200 deaths
(40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer
ranks second among cancer deaths in women. According to the most
recent data, mortality rates declined significantly during
1992-1996 with the largest decreases in younger women, both white
and black. These decreases were probably the result of earlier
detection and improved treatment.
[0022] Taking into account the medical circumstances and the
patient's preferences, treatment of breast cancer may involve
lumpectomy (local removal of the tumor) and removal of the lymph
nodes under the arm; mastectomy (surgical removal of the breast)
and removal of the lymph nodes under the arm; radiation therapy;
chemotherapy; or hormone therapy. Often, two or more methods are
used in combination. Numerous studies have shown that, for early
stage disease, long-term survival rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical
mastectomy. Significant advances in reconstruction techniques
provide several options for breast reconstruction after mastectomy.
Recently, such reconstruction has been done at the same time as the
mastectomy.
[0023] Local excision of ductal carcinoma in situ (DCIS) with
adequate amounts of surrounding normal breast tissue may prevent
the local recurrence of the DCIS. Radiation to the breast and/or
tamoxifen may reduce the chance of DCIS occurring in the remaining
breast tissue. This is important because DCIS, if left untreated,
may develop into invasive breast cancer. Nevertheless, there are
serious side effects or sequelae to these treatments. There is,
therefore, a need for efficacious breast cancer treatments.
[0024] There were an estimated 23,100 new cases of ovarian cancer
in the United States in 2000. It accounts for 4% of all cancers
among women and ranks second among gynecologic cancers. During
1992-1996, ovarian cancer incidence rates were significantly
declining. Consequent to ovarian cancer, there were an estimated
14,000 deaths in 2000. Ovarian cancer causes more deaths than any
other cancer of the female reproductive system.
[0025] Surgery, radiation therapy, and chemotherapy are treatment
options for ovarian cancer. Surgery usually includes the removal of
one or both ovaries, the fallopian tubes (salpingo-oophorectomy),
and the uterus (hysterectomy). In some very early tumors, only the
involved ovary will be removed, especially in young women who wish
to have children. In advanced disease, an attempt is made to remove
all intra-abdominal disease to enhance the effect of chemotherapy.
There continues to be an important need for effective treatment
options for ovarian cancer.
[0026] There were an estimated 28,300 new cases of pancreatic
cancer in the United States in 2000. Over the past 20 years, rates
of pancreatic cancer have declined in men. Rates among women have
remained approximately constant but may be beginning to decline.
Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the
United States. Over the past 20 years, there has been a slight but
significant decrease in mortality rates among men (about -0.9% per
year) while rates have increased slightly among women.
[0027] Surgery, radiation therapy, and chemotherapy are treatment
options for pancreatic cancer. These treatment options can extend
survival and/or relieve symptoms in many patients but are not
likely to produce a cure for most. There is a significant need for
additional therapeutic and diagnostic options for pancreatic
cancer.
SUMMARY OF THE INVENTION
[0028] The present invention relates to a novel nucleic acid
sequence and its encoded polypeptide, designated 158P1H4. As used
herein, "158P1H4" can refer to the novel polynucleotides and/or
polypeptides or both of the disclosed invention. As used herein
158P1H4 also refers to the novel polynucleotides and/or
polypeptides of 158P1F4 as disclosed herein unless the context
clearly indicates otherwise.
[0029] Nucleic acids encoding 158P1H4 are over-expressed in the
cancer(s) listed in Table I. Northern blot expression analysis of
158P1H4 expression in normal tissues shows a restricted expression
pattern in adult tissues. The nucleotide (FIG. 2) and amino acid
(FIG. 2, and FIG. 3) sequences of 158P1H4 are provided. The
tissue-related profile of 158P1H4 in normal adult tissues, combined
with the over-expression observed in bladder tumors, shows that
158P1H4 is aberrantly over-expressed in at least some cancers.
Thus, 158P1H4 nucleic acids and polypeptides serve as a useful
diagnostic agent (or indicator) and/or therapeutic target for
cancers of the tissues, such as those listed in Table I.
[0030] The invention provides polynucleotides corresponding or
complementary to all or part of the 158P1H4 nucleic acids, mRNAs,
and/or coding sequences, preferably in isolated form, including
polynucleotides encoding 158P1H4-related proteins and fragments of
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, or more than 25 contiguous amino acids; at least
about 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or
more than 100 contiguous amino acids of a 158P1H4-related protein,
as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA
hybrids, and related molecules (such as PNAs), polynucleotides or
oligonucleotides complementary or having at least a 90% homology to
158P1H4 nucleic acid sequences or mRNA sequences or parts thereof,
and polynucleotides or oligonucleotides that hybridize to the
158P1H4 genes, mRNAs, or to 158P1H4-encoding polynucleotides. Also
provided are means for isolating cDNAs and the gene(s) encoding
158P1H4. Recombinant DNA molecules containing 158P 1H4
polynucleotides, cells transformed or transduced with such
molecules, and host-vector systems for the expression of 158P1H4
gene products are also provided. The invention further provides
antibodies that bind to 158P1H4 proteins and polypeptide fragments
thereof, including polyclonal and monoclonal antibodies, murine and
other mammalian antibodies, chimeric antibodies, humanized and
fully human antibodies, and antibodies labeled with a detectable
marker. The invention also comprises T cell clones that recognize
an epitope of 158P1H4 in the context of a particular HLA
molecule.
[0031] The invention further provides methods for detecting the
presence, amount, and status of 158P1H4 polynucleotides and
proteins in various biological samples, as well as methods for
identifying cells that express 158P1H4 polynucleotides and
polypeptides. A typical embodiment of this invention provides
methods for monitoring 158P1H4 polynucleotides and polypeptides in
a tissue or hematology sample having or suspected of having some
form of growth dysregulation such as cancer.
[0032] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 158P1H4 such as bladder cancers, including therapies aimed
at inhibiting the transcription, translation, processing or
function of 158P1H4 as well as cancer vaccines.
BRIEF DESCRIPTION OF THE FIGURES
[0033] FIG. 1. 158P1H4 SSH nucleic acid sequence. The 158P1H4 SSH
sequence contains 90 bp. (SEQ ID. NO.:701)
[0034] FIG. 2. The cDNA (SEQ ID. NO.:702) and amino acid (SEQ ID.
NO.:703) sequences of 158P1H4. The start methionine is underlined.
The open reading frame extends from nucleic acid 175 to 1569
including the stop codon.
[0035] FIG. 3. Amino acid sequence of 158P1H4 (SEQ ID. NO.:703).
The 158P1H4 protein has 464 amino acids with calculated molecular
weight of 54.1 kDa, and pI of 6.5. 158P1H4 is predicted to be a
nuclear protein (56%), with a lower possibility of mitochondrial
localization (21.7%).
[0036] FIG. 4, panels A-C. Sequence alignment of 158P1H4 with mouse
gene GenBank accession AK014536 (SEQ ID. NOS.:704-6).
[0037] FIG. 5a. Amino acid sequence alignment of 158P1H4 with mouse
protein (SEQ ID. NO.:707, corresponding to GenBank Accession
BAB29419.1).
[0038] FIG. 5b. Amino acid sequence alignment of 158P1H4 with human
sorting nexin 17 (SEQ ID. NO.:708).
[0039] FIG. 6. RT-PCR analysis of 158P1H4 expression. First strand
cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney),
viral pool 2 (VP2: pancreas, colon and stomach), LAPC xenograft
pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC9AI), prostate cancer
pool, and bladder cancer pool. Normalization was performed by PCR
using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 158P1H4, was performed at 26 and 30 cycles of
amplification. Expression of 158P1H4 is observed in bladder cancer
pool and in the metastasis pool but not in normal tissues.
[0040] FIG. 7. Expression of 158P1H4 in normal human tissues by
Northern blot analysis. (A) and (B) Two multiple tissue Northern
blots with 2 .mu.g of mRNA/lane, were probed with the 158P1H4 probe
defined in FIG. 18A. Size standards in kilobases (kb) are indicated
on the side. The results show undetectable levels of 158P1H4
expression in all 16 human normal tissues tested.
[0041] FIG. 8. Expression of 158P1H4 in bladder cancer patient
samples. RNA was extracted from bladder cancer cell lines (CL),
normal bladder (Nb), bladder tumors (T) and their normal adjacent
tissues (N) derived from bladder cancer patients. Northern blots
with 10 .mu.g of total RNA/lane were probed with the 158P1H4 probe
defined in FIG. 18A. Size standards in kilobases (kb) are indicated
on the side. The results show expression of 158P1H4 in 3 bladder
tumors tested and, to a much lower extent, in one normal adjacent
tissue.
[0042] FIG. 9. Hydrophilicity amino acid profile of 158P1H4
determined by computer algorithm sequence analysis using the method
of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad.
Sci. U.S.A. 78:3824-3828) accessed on the Protscale website
(www.expasy.ch/cgi-bin/pr- otscale.p1) through the ExPasy molecular
biology server.
[0043] FIG. 10. Hydropathicity amino acid profile of 158P1H4
determined by computer algorithm sequence analysis using the method
of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol.
Biol. 157:105-132) accessed on the ProtScale website
(www.expasy.ch/cgi-bin/protscale.p1) through the ExPasy molecular
biology server.
[0044] FIG. 11. Percent accessible residues amino acid profile of
158P1H4 determined by computer algorithm sequence analysis using
the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on
the ProtScale website (www.expasy.ch/cgi-bin/protscale.p1) through
the ExPasy molecular biology server.
[0045] FIG. 12. Average flexibility amino acid profile of 158P1H4
determined by computer algorithm sequence analysis using the method
of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K.,
1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the
ProtScale website (www.expasy.ch/cgi-bin/protscale.p1) through the
ExPasy molecular biology server.
[0046] FIG. 13. Beta-turn amino acid profile of 158P1H4 determined
by computer algorithm sequence analysis using the method of Deleage
and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294)
accessed on the ProtScale website
(www.expasy.ch/cgi-bin/protscale.p1) through the ExPasy molecular
biology server.
[0047] FIG. 14. 158P1F4 SSH nucleic acid sequence. The 158P1F4 SSH
sequence contains 214 bp. (SEQ ID. NO.:725)
[0048] FIG. 15. RT-PCR analysis of 158P1F4 expression. First strand
cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney),
vital pool 2 (VP2, pancreas, colon and stomach), and bladder cancer
pool. Normalization was performed by PCR using primers to actin and
GAPDH. Semi-quantitative PCR, using primers to 158P1F4, was
performed at 26 and 30 cycles of amplification. Expression of
158P1F4 is observed in bladder cancer pool but not in any normal
tissues tested.
[0049] FIG. 16. Expression of 158P1F4 in normal human tissues by
Northern blot analysis. Two multiple tissue northern blots, with 2
.mu.g of mRNA/lane, were probed with the 158P1F4 SSH fragment. Size
standards in kilobases (kb) are indicated on the side. The results
show undetectable levels of 158P1F4 expression in all 16 human
normal tissues tested.
[0050] FIG. 17. Expression of 158P1F4 in bladder cancer patient
samples. RNA was extracted from a pool of 3 bladder cancer tumors
derived from bladder cancer patients (BCP), and from normal
prostate (NP), bladder (NB), and kidney (NK). Northern blots with
10 .mu.g of total RNA/lane were probed with the 158P1F4 SSH
sequences. Size standards in kilobases (kb) are indicated on the
side. The results show expression of 158P1F4 in the the bladder
cancer pool but not in the normal tissues.
[0051] FIG. 18A. Schematic Diagram of 158P1H4 and its Splice
Variants. Diagram shows nucleic acid sequences shared between
158P1H4 and its two splice variants. The location of the 158P1H4
and 158P4B5 SSH sequences are shown. Sequences used as probe 1 and
2 of 158 P4B5 and the 158P1H4 probe are indicated. Hatched boxes
indicate sequences present in variant 1 and/or variant 2 as
described in the annotation. The 5' and 3' termini of splice
variant 1 and splice variant 2 are the same. The 5' terminus is at
position 325 of 158P1H4 sequence, whereas the 3' terminus is from a
new exon not present in 158P1H4.
[0052] FIG. 18B. Sequences of 158P4B5 SSH fragment. The 241 base
pairs of 158P4B5 SSH nucleic acid sequence, which comprises probe
2, is shown. Sequence of 158P4B5 probe 1 is underlined.
[0053] FIG. 18C. Expression of 158P4B5 probe 1 in normal human
tissues. Two multiple tissue Northern blots, with 2 mg of
mRNA/lane, were probed with the 158P4B5 probe 1 sequence as defined
in FIGS. 18A and 18B. Size standards in kilobases (kb) are
indicated on the side. The results show that 158P4B5 probe 1
detected expression of multiple size transcripts in a tissue
specific pattern. Most prominent transcripts of 2 kb and 1 kb are
detected mostly in heart and skeletal muscle. Lower expression
levels are detected in other tissues.
[0054] FIG. 18D. Expression of 158P4B5 in bladder cancer patient
specimens detected using 158P4B5 probe 1. RNA was extracted from
normal bladder (Nb), bladder tumors (T) and their matched normal
adjacent tissue (NAT) isolated from bladder cancer patients.
Northern blots with 10 mg of total RNA/lane were hybridized with
probe 1 of 158P4B5 as defined in FIG. 18A and FIG. 18B. 158P4B5
probe 1 overlaps by 92 bp with sequences of splice variants 1 and
2. Size standards in kilobases (kb) are indicated on the side. The
arrow indicates transcripts similar in size to the transcript
detected using 158P1H4 probe. The results show expression of
multiple transcripts in bladder tumor and normal tissues. In
particular, an approximately 4.0 kb transcript is detected in 2 of
4 bladder tumors but not in normal tissues. These results indicate
that different size transcripts, which share exons with 158P1H4,
are expressed in bladder cancer patient specimens. Those can be
defined as splice variants of 158P1H4.
[0055] FIG. 18E. Expression of 158P4B5 in bladder cancer patient
specimens detected using 158P4B5 probe 2. RNA was extracted from
normal bladder (Nb), bladder tumors (T) and their matched normal
adjacent tissues (NAT) isolated from bladder cancer patients.
Northern blots with 10 mg of total RNA/lane were hybridized with
probe 2 of 158P4B5 as defined in FIG. 18A and FIG. 18B. 158P4B5
probe 2 overlaps by 64 bp with sequences of splice variants 1 and
2. Size standards in kilobases (kb) are indicated on the side. The
results show expression of a predominant 4 kb transcript in bladder
cancer tissues.
[0056] FIGS. 19A and 19B. Schematic representation and alignment at
the amino acid level of splice variant 1 (FIG. 19A) and splice
variant 2 (FIG. 19B) to 158P1H4.
[0057] FIG. 20. Alternative alignment at the amino acid level of
splice variants 1 and 2.
DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
[0058] I.) Definitions
[0059] II.) 158P1H4 Polynucleotides
[0060] II.A.) Uses of 158P1H4 Polynucleotides
[0061] II.A.1) Monitoring of Genetic Abnormalities
[0062] II.A.2.) Antisense Embodiments
[0063] II.A.3.) Primers and Primer Pairs
[0064] II.A.4.) Isolation of 158P1H4-Encoding Nucleic Acid
Molecules
[0065] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0066] III.) 158P1H4-related Proteins
[0067] III.A.) Motif-bearing Protein Embodiments
[0068] III.B.) Expression of 158P1H4-related Proteins
[0069] III.C.) Modifications of 158P1H4-related Proteins
[0070] III.D.) Uses of 158P1H4-related Proteins
[0071] IV.) 158P1H4 Antibodies
[0072] V.) 158P1H4 Cellular Immune Responses
[0073] VI.) 158P1H4 Transgenic Animals
[0074] VII.) Methods for the Detection of 158P1H4
[0075] VIII.) Methods for Monitoring the Status of 158P1H4-related
Genes and Their Products
[0076] IX.) Identification of Molecules That Interact With
158P1H4
[0077] X.) Therapeutic Methods and Compositions
[0078] X.A.) Anti-Cancer Vaccines
[0079] X.B.) 158P1H4 as a Target for Antibody-Based Therapy
[0080] X.C.) 158P1H4 as a Target for Cellular Immune Responses
[0081] X.C.1. Minigene Vaccines
[0082] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0083] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0084] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0085] X.D.) Adoptive Immunotherapy
[0086] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0087] XI.) Diagnostic and Prognostic Embodiments of 158P1H4.
[0088]
[0089] XII.) Inhibition of 158P1H4 Protein Function
[0090] XII.A.) Inhibition of 158P1H4 With Intracellular
Antibodies
[0091] XII.B.) Inhibition of 158P1H4 with Recombinant Proteins
[0092] XII.C.) Inhibition of 158P1H4 Transcription or
Translation
[0093] XII.D.) General Considerations for Therapeutic
Strategies
[0094] XIII.) KITS
[0095] I.) Definitions
[0096] Unless otherwise defined, all terms of art, notations and
other scientific terms or terminology used herein are intended to
have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference, and the inclusion of such definitions herein
should not necessarily be construed to represent a substantial
difference over what is generally understood in the art. Many of
the techniques and procedures described or referenced herein are
well understood and commonly employed using conventional
methodology by those skilled in the art, such as, for example, the
widely utilized molecular cloning methodologies described in
Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd.
edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. As appropriate, procedures involving the use of
commercially available kits and reagents are generally carried out
in accordance with manufacturer defined protocols and/or parameters
unless otherwise noted.
[0097] As used herein 158P1H4 also refers to the novel
polynucleotides and/or polypeptides of 158P1F4 as disclosed herein
unless the context clearly indicates otherwise to one of ordinary
skill in the art.
[0098] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 158P1H4 (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence 158P1H4. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0099] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 158P1H4-related protein). For example an analog of
the 158P1H4 protein can be specifically bound by an antibody or T
cell that specifically binds to 158P1H4 protein.
[0100] The term "antibody" is used in the broadest sense. Therefore
an "antibody" can be naturally occurring or man-made such as
monoclonal antibodies produced by conventional hybridoma
technology. Anti-158P1H4 antibodies bind 158P1H4 proteins, or a
fragment thereof, and comprise monoclonal and polyclonal antibodies
as well as fragments containing the antigen-binding domain and/or
one or more complementarity determining regions of these
antibodies.
[0101] An "antibody fragment" is defined as at least a portion of
the variable region of the immunoglobulin molecule that binds to
its target, i.e., the antigen-binding region. In one embodiment it
specifically covers single anti-158P1H4 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-158P1H4 antibody compositions with polyepitopic
specificity.
[0102] The term "codon optimized sequences" refers to nucleotide
sequences that have been optimized for a particular host species by
replacing any one or more than one codon having a usage frequency
of less than about 20%, more preferably less than about 30% or 40%.
A sequence may be "completely optimized"to contain no codon having
a usage frequency of less than about 20%, more preferably less than
about 30% or 40%. Nucleotide sequences that have been optimized for
expression in a given host species by elimination of spurious
polyadenylation sequences, elimination of exon/intron splicing
signals, elimination of transposon-like repeats and/or optimization
of GC content in addition to codon optimization are referred to
herein as an "expression enhanced sequences."
[0103] The term "cytotoxic agent" refers to a substance that
inhibits or prevents one or more than one function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes chemotherapeutic agents, and toxins such as
small molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants
thereof. Examples of cytotoxic agents include, but are not limited
to maytansinoids, yttrium, bismuth, ricin, ricin A-chain,
doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicine,
dihydroxy anthracin dione, actinomycin, diphtheria toxin,
Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A
chain, alpha-sarcin, gelonin, mitogellin, retstrictocin,
phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria
officinalis inhibitor, and glucocorticoid and other
chemotherapeutic agents, as well as radioisotopes such as
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of Lu.
Antibodies may also be conjugated to an anti-cancer pro-drug
activating enzyme capable of converting the pro-drug to its active
form.
[0104] The term "homolog" refers to a molecule which exhibits
homology to another molecule, by for example, having sequences of
chemical residues that are the same or similar at corresponding
positions.
[0105] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see, e.g.,
Stites, et al, IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos,
Calif. (1994).
[0106] The terms "hybridize", "hybridizing", "hybridizes"and the
like, used in the context of polynucleotides, are meant to refer to
conventional hybridization conditions, preferably such as
hybridization in 50% formamide/6.times.SSC/0.1% SDS/100 .mu.g/ml
ssDNA, in which temperatures for hybridization are above 37 degrees
C. and temperatures for washing in 0.1.times.SSC/0.1% SDS are above
55 degrees C.
[0107] The terms "invasive bladder cancer" means bladder cancers
that have extended into the bladder muscle wall, and are meant to
include stage stage T2-T4 and disease under the TNM (tumor, node,
metastasis) system. In general, these patients have substantially
less favorable outcomes compared to patients having non-invasive
cancer. Following cystectomy, 50% or more of the patients with
invasive cancer will develop metastasis (Whittmore. Semin Urol
1983; 1:4-10).
[0108] The phrases "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated, or
present, with the peptides in their in situ environment. For
example, a polynucleotide is said to be "isolated" when it is
substantially separated from contaminant polynucleotides that
correspond or are complementary to nucleic acids other than those
of 158P1H4 or that encode polypeptides other than 158P1H4 gene
product or fragments thereof. A skilled artisan can readily employ
nucleic acid isolation procedures to obtain an isolated 158P1H4
polynucleotide. A protein is said to be "isolated," for example,
when physical, mechanical and/or chemical methods are employed to
remove the 158P1H4 protein from cellular constituents that are
normally associated, or present, with the protein. A skilled
artisan can readily employ standard purification methods to obtain
an isolated 158P1H4 protein. Alternatively, an isolated protein can
be prepared by synthetic or chemical means.
[0109] The term "mammal" refers to any organism classified as a
mammal, including mice, rats, rabbits, dogs, cats, cows, horses and
humans. In one embodiment of the invention, the mammal is a mouse.
In another embodiment of the invention, the mammal is a human.
[0110] The terms "metastatic bladder cancer" and "metastatic
disease" mean bladder cancers that have spread to regional lymph
nodes or to distant sites, and are meant to stage
T.times.N.times.M+ under the TNM system. The most common site for
bladder cancer metastasis is lymph node. Other common sites for
metastasis include lung, bone and liver.
[0111] The term "monoclonal antibody" refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the antibodies comprising the population are identical except
for possible naturally occurring mutations that are present in
minor amounts.
[0112] A "motif", as in biological motif of an 158P1H4-related
protein, refers to any pattern of amino acids forming part of the
primary sequence of a protein, that is associated with a particular
function (e.g. protein-protein interaction, protein-DNA
interaction, etc) or modification (e.g. that is phosphorylated,
glycosylated or amidated), or localization (e.g. secretory
sequence, nuclear localization sequence, etc.) or a sequence that
is correlated with being immunogenic, either humorally or
cellularly. A motif can be either contiguous or capable of being
aligned to certain positions that are generally correlated with a
certain function or property. In the context of HLA motifs, "motif"
refers to the pattern of residues in a peptide of defined length,
usually a peptide of from about 8 to about 13 amino acids for a
class I HLA motif and from about 6 to about 25 amino acids for a
class II HLA motif, which is recognized by a particular HLA
molecule. Peptide motifs for HLA binding are typically different
for each protein encoded by each human HLA allele and differ in the
pattern of the primary and secondary anchor residues.
[0113] A "pharmaceutical excipient" comprises a material such as an
adjuvant, a carrier, pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservative, and the like.
[0114] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with mammals,
such as humans.
[0115] The term "polynucleotide" means a polymeric form of
nucleotides of at least 3, 4, 5, 6, 7, 8, 9, or 10 bases or base
pairs in length, either ribonucleotides or deoxynucleotides or a
modified form of either type of nucleotide, and is meant to include
single and double stranded forms of DNA and/or RNA. In the art,
this term is often used interchangeably with "oligonucleotide",
although "oligonucleotide" may be used to refer to the subset of
polynucleotides less than about 50 nucleotides in length. A
polynucleotide can comprise a nucleotide sequence disclosed herein
wherein thymidine (T) (as shown for example in SEQ ID NO: 702) can
also be uracil (U); this definition pertains to the differences
between the chemical structures of DNA and RNA, in particular the
observation that one of the four major bases in RNA is uracil (U)
instead of thymidine (T).
[0116] The term "polypeptide" means a polymer of at least about 4,
5, 6, 7, or 8 amino acids. Throughout the specification, standard
three letter or single letter designations for amino acids are
used. In the art, this term is often used interchangeably with
"peptide" or "protein", thus "peptide" may be used to refer to the
subset of polypeptides less than about 50 amino acids in
length.
[0117] An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the HLA
molecule. One to three, usually two, primary anchor residues within
a peptide of defined length generally defines a "motif" for an
immunogenic peptide. These residues are understood to fit in close
contact with peptide binding groove of an HLA molecule, with their
side chains buried in specific pockets of the binding groove. In
one embodiment, for example, the primary anchor residues for an HLA
class I molecule are located at position 2 (from the amino terminal
position) and at the carboxyl terminal position of a 8, 9, 10, 11,
or 12 residue peptide epitope in accordance with the invention. In
another embodiment, for example, the primary anchor residues of a
peptide that will bind an HLA class II molecule are spaced relative
to each other, rather than to the termini of a peptide, where the
peptide is generally of at least 9 amino acids in length. The
primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the
primary and/or secondary anchor positions shown in Table IV. Such
analogs are used to modulate the binding affinity and/or population
coverage of a peptide comprising a particular HLA motif or
supermotif.
[0118] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0119] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured nucleic acid sequences to reanneal when
complementary strands are present in an environment below their
melting temperature. The higher the degree of desired homology
between the probe and hybridizable sequence, the higher the
relative temperature that can be used. As a result, it follows that
higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For
additional details and explanation of stringency of hybridization
reactions, see Ausubel et al., Current Protocols in Molecular
Biology, Wiley Interscience Publishers, (1995).
[0120] "Stringent conditions" or "high stringency conditions", as
defined herein, are identified by, but not limited to, those that:
(1) employ low ionic strength and high temperature for washing, for
example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium
dodecyl sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium. citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C. "Moderately stringent conditions"
are described by, but not limited to, those in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, New York: Cold Spring
Harbor Press, 1989, and include the use of washing solution and
hybridization conditions (e.g., temperature, ionic strength and
%SDS) less stringent than those described above. An example of
moderately stringent conditions is overnight incubation at
37.degree. C. in a solution comprising: 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times.Denhardt's solution, 10% dextran sulfate, and 20
mg/mL denatured sheared salmon sperm DNA, followed by washing the
filters in 1.times.SSC at about 37-50.degree. C. The skilled
artisan will recognize how to adjust the temperature, ionic
strength, etc. as necessary to accommodate factors such as probe
length and the like.
[0121] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles.
[0122] A "transgenic animal" (e.g., a mouse or rat) is an animal
having cells that contain a transgene, which transgene was
introduced into the animal or an ancestor of the animal at a
prenatal, e.g., an embryonic stage. A "transgene" is a DNA that is
integrated into the genome of a cell from which a transgenic animal
develops.
[0123] As used herein, an HLA or cellular immune response "vaccine"
is a composition that contains or encodes one or more peptides of
the invention. There are numerous embodiments of such vaccines,
such as a cocktail of one or more individual peptides; one or more
peptides of the invention comprised by a polyepitopic peptide; or
nucleic acids that encode such individual peptides or polypeptides,
e.g., a minigene that encodes a polyepitopic peptide. The "one or
more peptides" can include any whole unit integer from 1-150 or
more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, or 150 or more peptides of the
invention. The peptides or polypeptides can optionally be modified,
such as by lipidation, addition of targeting or other sequences.
HLA class I peptides of the invention can be admixed with, or
linked to, HLA class II peptides, to facilitate activation of both
cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can
also comprise peptide-pulsed antigen presenting cells, e.g.,
dendritic cells.
[0124] The term "variant" refers to a molecule that exhibits a
variation from a described type or norm, such as a protein that has
one or more different amino acid residues in the corresponding
position(s) of a specifically described protein (e.g. the 158P1H4
protein shown in FIG. 2 or FIG. 3). An analog is an example of a
variant protein.
[0125] The 158P1H4-related proteins of the invention include those
specifically identified herein, as well as allelic variants,
conservative substitution variants, analogs and homologs that can
be isolated/generated and characterized without undue
experimentation following the methods outlined herein or readily
available in the art. Fusion proteins that combine parts of
different 158P1H4 proteins or fragments thereof, as well as fusion
proteins of a 158P1H4 protein and a heterologous polypeptide are
also included. Such 158P1H4 proteins are collectively referred to
as the 158P1H4-related proteins, the proteins of the invention, or
158P1H4. The term "158P1H4-related protein" refers to a polypeptide
fragment or an 158P1H4 protein sequence of 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more
than 25 amino acids; or, at least about 30, 35, 40, 45, 50, 55, 60,
65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.
[0126] II.) 158P1H4 Polynucleotides
[0127] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of an 158P1H4 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding an 158P1H4-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to an 158P1H4
gene or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to an 158P1H4 gene, mRNA, or to an
158P1H4 encoding polynucleotide (collectively, "158P1H4
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0128] Embodiments of a 158P1H4 polynucleotide include: a 158P1H4
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 158P1H4 as shown in FIG. 2, wherein T is U; at least 10
contiguous nucleotides of a polynucleotide having the sequence as
shown in FIG. 2; or, at least 10 contiguous nucleotides of a
polynucleotide having the sequence as shown in FIG. 2 where T is U.
For example, embodiments of 158P1H4 nucleotides comprise, without
limitation:
[0129] (a) a polynucleotide comprising or consisting of the
sequence as shown in FIG. 2 (SEQ ID NO.:702), wherein T can also be
U;
[0130] (b) a polynucleotide comprising or consisting of the
sequence as shown in FIG. 2 (SEQ ID NO.:702), from nucleotide
residue number 4 through nucleotide residue number 1386, wherein T
can also be U;
[0131] (c) a polynucleotide that encodes a 158P1H4-related protein
whose sequence is encoded by the cDNAs contained in the plasmid
designated p158P1H4-EBB10 deposited with American Type Culture
Collection as Accession No. PTA-3136;
[0132] (d) a polynucleotide that encodes an 158P1H4-related protein
that is at least 90% homologous to the entire amino acid sequence
shown in SEQ ID NO.:703;
[0133] (e) a polynucleotide that encodes an 158P1H4-related protein
that is at least 90% identical to the entire amino acid sequence
shown in SEQ ID NO: 703;
[0134] (f) a polynucleotide that encodes at least one peptide set
forth in Tables V-XVIII;
[0135] (g) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
440 that includes an amino acid position having a value greater
than 0.5 in the Hydrophilicity profile of FIG. 9;
[0136] (h) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
440 that includes an amino acid position having a value less than
0.5 in the Hydropathicity profile of FIG. 10;
[0137] (i) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
440 that includes an amino acid position having a value greater
than 0.5 in the Percent Accessible Residues profile of FIG. 11;
[0138] (j) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
440 that includes an amino acid position having a value greater
than 0.5 in the Average Flexibility profile on FIG. 12;
[0139] (k) a polynucleotide that encodes a peptide region of at
least 5 amino acids of FIG. 3 in any whole number increment up to
440 that includes an amino acid position having a value greater
than 0.5 in the Beta-turn profile of FIG. 13;
[0140] (l) a polynucleotide that is fully complementary to a
polynucleotide of any one of (a)-(k);
[0141] (m) a polynucleotide that selectively hybridizes under
stringent conditions to a polynucleotide of (a)-(l); and
[0142] (n) a polynucleotide of any of (a)-(m) or peptide of (o)
(see immediately below) together with a pharmaceutical excipient
and/or in a human unit dose form.
[0143] Regarding item (n) immediately above, examples of
embodiments of 158P1H4 polypeptides comprise, without
limitation:
[0144] (o) a peptide that is encoded by any of (a)-(k).
[0145] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0146] Typical embodiments of the invention disclosed herein
include 158P1H4 polynucleotides that encode specific portions of
the 158P1H4 mRNA sequence (and those which are complementary to
such sequences) such as those that encode the protein and fragments
thereof, for example of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17,18, 19,20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, or 440 contiguous amino acids.
[0147] For example, representative embodiments of the invention
disclosed herein include: polynucleotides and their encoded
peptides themselves encoding about amino acid 1 to about amino acid
10 of the 158P1H4 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 158P1H4 protein shown in FIG. 2, or FIG. 3, polynucleotides
encoding about amino acid 20 to about amino acid 30 of the 158P1H4
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 158P1H4 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 158P1H4 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 158P1H4 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 158P1H4 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 158P1H4
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 158P1H4 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 158P1H4 protein shown in FIG. 2 or
FIG. 3, in increments of about 10 amino acids, ending at the
carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3.
Accordingly polynucleotides encoding portions of the amino acid
sequence (of about 10 amino acids), of amino acids 100 through the
carboxyl terminal amino acid of the 158P1H4 protein are embodiments
of the invention. Wherein it is understood that each particular
amino acid position discloses that position plus or minus five
amino acid residues.
[0148] Polynucleotides encoding relatively long portions of the
158P1H4 protein are also within the scope of the invention. For
example, polynucleotides encoding from about amino acid 1 (or 20 or
30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of
the 158P1H4 protein shown in FIG. 2 or FIG can be generated by a
variety of techniques well known in the art. These polynucleotide
fragments can include any portion of the 158P1H4 sequence as shown
in FIG. 2 or FIG. 3.
[0149] Additional illustrative embodiments of the invention
disclosed herein include 158P1H4 polynucleotide fragments encoding
one or more of the biological motifs contained within the 158P1H4
protein sequence, including one or more of the motif-bearing
subsequences of the 158P1H4 protein set forth in Tables V-XVIII. In
another embodiment, typical polynucleotide fragments of the
invention encode one or more of the regions of 158P1H4 that exhibit
homology to a known molecule. In another embodiment of the
invention, typical polynucleotide fragments can encode one or more
of the 158P1H4 N-glycosylation sites, cAMP and cGMP-dependent
protein kinase phosphorylation sites, casein kinase II
phosphorylation sites or N-myristoylation site and amidation
sites.
[0150] II.A.) Uses of 158P1H4 Polynucleotides
[0151] II.A.1.) Monitoring of Genetic Abnormalities
[0152] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 158P1H4 gene maps to
the chromosomal location set forth in Example 3. For example,
because the 158P1H4 gene maps to this chromosome, polynucleotides
that encode different regions of the 158P1H4 protein are used to
characterize cytogenetic abnormalities of this chromosomal locale,
such as abnormalities that are identified as being associated with
various cancers. In certain genes, a variety of chromosomal
abnormalities including rearrangements have been identified as
frequent cytogenetic abnormalities in a number of different cancers
(see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);
Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,
P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding
specific regions of the 158P1H4 protein provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 158P1H4 that may contribute to the malignant phenotype. In
this context, these polynucleotides satisfy a need in the art for
expanding the sensitivity of chromosomal screening in order to
identify more subtle and less common chromosomal abnormalities (see
e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057
(1994)).
[0153] Furthermore, as 158P1H4 was shown to be highly expressed in
bladder and other cancers, 158P1H4 polynucleotides are used in
methods assessing the status of 158P1H4 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 158P1H4 protein are used to assess the
presence of perturbations (such as deletions, insertions, point
mutations, or alterations resulting in a loss of an antigen etc.)
in specific regions of the 158P1H4 gene, such as such regions
containing one or more motifs. Exemplary assays include both RT-PCR
assays as well as single-strand conformation polymorphism (SSCP)
analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8):
369-378 (1999), both of which utilize polynucleotides encoding
specific regions of a protein to examine these regions within the
protein.
[0154] II.A.2.) Antisense Embodiments
[0155] Other specifically contemplated nucleic acid related
embodiments of the invention disclosed herein are genomic DNA,
cDNAs, ribozymes, and antisense molecules, as well as nucleic acid
molecules based on an alternative backbone, or including
alternative bases, whether derived from natural sources or
synthesized, and include molecules capable of inhibiting the RNA or
protein expression of 158P1H4. For example, antisense molecules can
be RNAs or other molecules, including peptide nucleic acids (PNAs)
or non-nucleic acid molecules such as phosphorothioate derivatives,
that specifically bind DNA or RNA in a base pair-dependent manner.
A skilled artisan can readily obtain these classes of nucleic acid
molecules using the 158P1H4 polynucleotides and polynucleotide
sequences disclosed herein.
[0156] Antisense technology entails the administration of exogenous
oligonucleotides that bind to a target polynucleotide located
within the cells. The term "antisense" refers to the fact that such
oligonucleotides are complementary to their intracellular targets,
e.g., 158P1H4. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 158P1H4 antisense oligonucleotides of
the present invention include derivatives such as
S-oligonucleotides (phosphorothioate derivatives or S-oligos, see,
Jack Cohen, supra), which exhibit enhanced cancer cell growth
inhibitory action. S-oligos (nucleoside phosphorothioates) are
isoelectronic analogs of an oligonucleotide (O-oligo) in which a
nonbridging oxygen atom of the phosphate group is replaced by a
sulfur atom. The S-oligos of the present invention can be prepared
by treatment of the corresponding O-oligos with
3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer
reagent. See Iyer, R. P. et al, J. Org. Chem. 55:4693-4698 (1990);
and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).
Additional 158P1H4 antisense oligonucleotides of the present
invention include morpholino antisense oligonucleotides known in
the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic
Acid Drug Development 6: 169-175).
[0157] The 158P1H4 antisense oligonucleotides of the present
invention typically can be RNA or DNA that is complementary to and
stably hybridizes with the first 100 5' codons or last 100 3'
codons of the 158P1H4 genomic sequence or the corresponding mRNA.
Absolute complementarity is not required, although high degrees of
complementarity are preferred. Use of an oligonucleotide
complementary to this region allows for the selective hybridization
to 158P1H4 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 158P1H4 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 158P1H4 mRNA. Optionally, 158P1H4 antisense
oligonucleotide is a 30-mer oligonucleotide that is complementary
to a region in the first 10 5' codons or last 10 3' codons of
158P1H4. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 158P1H4 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12:
510-515 (1996).
[0158] II.A3.) Primers and Primer Pairs
[0159] Further specific embodiments of this nucleotides of the
invention include primers and primer pairs, which allow the
specific amplification of polynucleotides of the invention or of
any specific parts thereof, and probes that selectively or
specifically hybridize to nucleic acid molecules of the invention
or to any part thereof. Primers may also be used as probes and can
be labeled with a detectable marker, such as, for example, a
radioisotope, fluorescent compound, bioluminescent compound, a
chemiluminescent compound, metal chelator or enzyme. Such probes
and primers are used to detect the presence of a 158P1H4
polynucleotide in a sample and as a means for detecting a cell
expressing a 158P1H4 protein.
[0160] Examples of such probes include polypeptides comprising all
or part of the human 158P1H4 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 158P1H4
mRNAs are also described in the Examples. As will be understood by
the skilled artisan, a great many different primers and probes can
be prepared based on the sequences provided herein and used
effectively to amplify and/or detect a 158P1H4 mRNA. Preferred
probes of the invention are polynucleotides of more than about 9,
about 12, about 15, about 18, about 20, about 23, about 25, about
30, about 35, about 40, about 50 consecutive nucleotides found in
158P1H4 nucleic acids disclosed herein.
[0161] The 158P1H4 polynucleotides of the invention are useful for
a variety of purposes, including but not limited to their use as
probes and primers for the amplification and/or detection of the
158P1H4 gene(s), mRNA(s), or fragments thereof; as reagents for the
diagnosis and/or prognosis of bladder cancer and other cancers; as
coding sequences capable of directing the expression of 158P1H4
polypeptides; as tools for modulating or inhibiting the expression
of the 158P1H4 gene(s) and/or translation of the 158P1H4
transcript(s); and as therapeutic agents.
[0162] II.A.4.) Isolation of 158P1H4-Encoding Nucleic Acid
Molecules
[0163] The 158P1H4 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 158P1H4 gene
product(s), as well as the isolation of polynucleotides encoding
158P1H4 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of the 158P1H4 gene product as
well as polynucleotides that encode analogs of 158P1H4-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding an 158P1H4 gene are well known
(see, for example, Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York,
1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,
Wiley and Sons, 1995). For example, lambda phage cloning
methodologies can be conveniently employed, using commercially
available cloning systems (e.g., Lambda ZAP Express, Stratagene).
Phage clones containing 158P1H4 gene cDNAs can be identified by
probing with a labeled 158P1H4 cDNA or a fragment thereof. For
example, in one embodiment, the 158P1H4 cDNA (FIG. 2) or a portion
thereof can be synthesized and used as a probe to retrieve
overlapping and full-length cDNAs corresponding to a 158P1H4 gene.
The 158P1H4 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 158P1H4
DNA probes or primers.
[0164] The present invention includes the use of any probe as
described herein to identify and isolate a 158P1H4 or 158P1H4
related nucleic acid sequence from a naturally occurring source,
such as humans or other mammals, as well as the isolated nucleic
acid sequence per se, which would comprise all or most of the
sequences found in the probe used.
[0165] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0166] The invention also provides recombinant DNA or RNA molecules
containing an 158P1H4 polynucleotide, a fragment, analog or
homologue thereof, including but not limited to phages, plasmids,
phagemids, cosmids, YACs, BACs, as well as various viral and
non-viral vectors well known in the art, and cells transformed or
transfected with such recombinant DNA or RNA molecules. Methods for
generating such molecules are well known (see, for example,
Sambrook et al, 1989, supra).The invention further provides a
host-vector system comprising a recombinant DNA molecule containing
a 158P1H4 polynucleotide, fragment, analog or homologue thereof
within a suitable prokaryotic or eukaryotic host cell. Examples of
suitable eukaryotic host cells include a yeast cell, a plant cell,
or an animal cell, such as a mammalian cell or an insect cell
(e.g., a baculovirus-infectible cell such as an Sf9 or HighFive
cell). Examples of suitable mammalian cells include various bladder
cancer cell lines such as SCaBER, UM-UC3, HT1376, RT4, T24,
TCC-SUP, J82 and SW780, other transfectable or transducible bladder
cancer cell lines, as well as a number of mammalian cells routinely
used for the expression of recombinant proteins (e.g., COS, CHO,
293, 293T cells). More particularly, a polynucleotide comprising
the coding sequence of 158P1H4 or a fragment, analog or homolog
thereof can be used to generate 158P1H4 proteins or fragments
thereof using any number of host-vector systems routinely used and
widely known in the art.
[0167] A wide range of host-vector systems suitable for the
expression of 158P1H4 proteins or fragments thereof are available,
see for example, Sambrook et al., 1989, supra; Current Protocols in
Molecular Biology, 1995, supra). Preferred vectors for mammalian
expression include but are not limited to pcDNA 3.1 myc-His-tag
(Invitrogen) and the retroviral vector pSR atkneo (Muller et al.,
1991, MCB 11: 1785). Using these expression vectors, 158P1H4 can be
expressed in several bladder cancer and non-bladder cell lines,
including for example SCaBER, UM-UC3, HT1376, RT4, T24, TCC-SUP,
J82 and SW780. The host-vector systems of the invention are useful
for the production of a 158P1H4 protein or fragment thereof. Such
host-vector systems can be employed to study the functional
properties of 158P1H4 and 158P1H4 mutations or analogs.
[0168] Recombinant human 158P1H4 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 158P1H4-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 158P1H4 or fragment, analog or homolog thereof, the
158P1H4 or related protein is expressed in the 293T cells, and the
recombinant 158P1H4 protein is isolated using standard purification
methods (e.g., affinity purification using anti-158P1H4
antibodies). In another embodiment, a 158P1H4 coding sequence is
subcloned into the retroviral vector pSR.alpha.MSVtkneo and used to
infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293
and rat-1 in order to establish 158P1H4 expressing cell lines.
Various other expression systems well known in the art can also be
employed. Expression constructs encoding a leader peptide joined in
frame to the 158P1H4 coding sequence can be used for the generation
of a secreted form of recombinant 158P1H4 protein.
[0169] As discussed herein, redundancy in the genetic code permits
variation in 158P1H4 gene sequences. In particular, it is known in
the art that specific host species often have specific codon
preferences, and thus one can adapt the disclosed sequence as
preferred for a desired host. For example, preferred analog codon
sequences typically have rare codons (i.e., codons having a usage
frequency of less than about 20% in known sequences of the desired
host) replaced with higher frequency codons. Codon preferences for
a specific species are calculated, for example, by utilizing codon
usage tables available on the INTERNET such as at URL
www.dna.affrc.go.jp/.about.nakamura/codon.html.
[0170] Additional sequence modifications are known to enhance
protein expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon/intron
splice site signals, transposon-like repeats, and/or other such
well-characterized sequences that are deleterious to gene
expression. The GC content of the sequence is adjusted to levels
average for a given cellular host, as calculated by reference to
known genes expressed in the host cell. Where possible, the
sequence is modified to avoid predicted hairpin secondary mRNA
structures. Other useful modifications include the addition of a
translational initiation consensus sequence at the start of the
open reading frame, as described in Kozak, Mol. Cell Biol.,
9:5073-5080 (1989). Skilled artisans understand that the general
rule that eukaryotic ribosomes initiate translation exclusively at
the 5' proximal AUG codon is abrogated only under rare conditions
(see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR
15(20): 8125-8148 (1987)).
[0171] III.) 158P1H4-related Proteins
[0172] Another aspect of the present invention provides
158P1H4-related proteins. Specific embodiments of 158P1H4 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 158P1H4 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 158P1H4 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 158P1H4 shown in FIG. 2 or FIG. 3.
[0173] In general, naturally occurring allelic variants of human
158P1H4 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of the
158P1H4 protein contain conservative amino acid substitutions
within the 158P1H4 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 158P1H4. One class of 158P1H4 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 158P1H4 amino acid sequence, but further
contain a radical departure from the sequence, such as a
non-conservative substitution, truncation, insertion or frame
shift. In comparisons of protein sequences, the terms, similarity,
identity, and homology each have a distinct meaning as appreciated
in the field of genetics. Moreover, orthology and paralogy can be
important concepts describing the relationship of members of a
given protein family in one organism to the members of the same
family in other organisms.
[0174] Amino acid abbreviations are provided in Table II.
Conservative amino acid substitutions can frequently be made in a
protein without altering either the conformation or the function of
the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more conservative
substitutions. Such changes include substituting any of isoleucine
(I), valine (V), and leucine (L) for any other of these hydrophobic
amino acids; aspartic acid (D) for glutamic acid (E) and vice
versa; glutamine (Q) for asparagine (N) and vice versa; and serine
(S) for threonine (T) and vice versa. Other substitutions can also
be considered conservative, depending on the environment of the
particular amino acid and its role in the three-dimensional
structure of the protein. For example, glycine (G) and alanine (A)
can frequently be interchangeable, as can alanine (A) and valine
(V). Methionine (M), which is relatively hydrophobic, can
frequently be interchanged with leucine and isoleucine, and
sometimes with valine. Lysine (K) and arginine (R) are frequently
interchangeable in locations in which the significant feature of
the amino acid residue is its charge and the differing pK's of
these two amino acid residues are not significant. Still other
changes can be considered "conservative" in particular environments
(see, e.g. Table III herein; pages 13-15 "Biochemistry" 2.sup.nd
ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS
1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;
270(20):11882-6).
[0175] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 158P1H4 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 158P1H4 variants can be made using methods known in
the art such as site-directed mutagenesis, alanine scanning, and
PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl.
Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487
(1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)),
restriction selection mutagenesis (Wells et al., Philos. Trans. R.
Soc. London SerA, 317:415 (1986)) or other known techniques can be
performed on the cloned DNA to produce the 158P1H4 variant DNA.
[0176] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence that
is involved in a specific biological activity such as a
protein-protein interaction. Among the preferred scanning amino
acids are relatively small, neutral amino acids. Such amino acids
include alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid among this group because
it eliminates the side-chain beyond the beta-carbon and is less
likely to alter the main-chain conformation of the variant. Alanine
is also typically preferred because it is the most common amino
acid. Further, it is frequently found in both buried and exposed
positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does
not yield adequate amounts of variant, an isosteric amino acid can
be used.
[0177] As defined herein, 158P1H4 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 158P1H4 protein having the amino
acid sequence of SEQ ID NO: 703. As used in this sentence, "cross
reactive" means that an antibody or T cell that specifically binds
to an 158P1H4 variant also specifically binds to the 158P1H4
protein having the amino acid sequence of SEQ ID NO: 703. A
polypeptide ceases to be a variant of the protein shown in SEQ ID
NO: 703 when it no longer contains any epitope capable of being
recognized by an antibody or T cell that specifically binds to the
158P1H4 protein. Those skilled in the art understand that
antibodies that recognize proteins bind to epitopes of varying
size, and a grouping of the order of about four or five amino
acids, contiguous or not, is regarded as a typical number of amino
acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000
165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73;
Schwartz et al., J Immunol (1985) 135(4):2598-608.
[0178] Another class of 158P1H4-related protein variants share 70%,
75%, 80%, 85% or 90% or more similarity with the amino acid
sequence of SEQ ID NO: 703 or a fragment thereof. Another specific
class of 158P1H14 protein variants or analogs comprise one or more
of the 158P1H4 biological motifs described herein or presently
known in the art. Thus, encompassed by the present invention are
analogs of 158P1H4 fragments (nucleic or amino acid) that have
altered functional (e.g. immunogenic) properties relative to the
starting fragment. It is to be appreciated that motifs now or which
become part of the art are to be applied to the nucleic or amino
acid sequences of FIG. 2 or FIG. 3.
[0179] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of the 158P1H4 protein shown in FIG. 2 or FIG. 3. For
example, representative embodiments of the invention comprise
peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15 or more contiguous amino acids of the 158P1H4 protein shown in
FIG. 2 or FIG. 3.
[0180] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid 1 to about amino acid 10 of the 158P1l H4 protein shown in
FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to
about amino acid 20 of the 158P1H14 protein shown in FIG. 2 or FIG.
3, polypeptides consisting of about amino acid 20 to about amino
acid 30 of the 158 P1H4 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 30 to about amino acid
40 of the 158P1H4 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 40 to about amino acid 50 of the
158P1H4 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 50 to about amino acid 60 of the 158P1H4
protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about
amino acid 60 to about amino acid 70 of the 158P1H4 protein shown
in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70
to about amino acid 80 of the 158P1H4 protein shown in FIG. 2 or
FIG. 3, polypeptides consisting of about amino acid 80 to about
amino acid 90 of the 158P1H4 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 90 to about amino acid
100 of the 158P1H4 protein shown in FIG. 2 or FIG. 3, etc.
throughout the entirety of the 158P1H4 amino acid sequence.
Moreover, polypeptides consisting of about amino acid 1 (or 20 or
30 or 40 etc.) to about amino acid 20, (or 130, or 140 or of the
158P1H4 protein shown in FIG. 2 or FIG. 3 are embodiments of the
invention. It is to be appreciated that the starting and stopping
positions in this paragraph refer to the specified position as well
as that position plus or minus 5 residues.
[0181] 158P1H4-related proteins are generated using standard
peptide synthesis technology or using chemical cleavage methods
well known in the art. Alternatively, recombinant methods can be
used to generate nucleic acid molecules that encode a
158P1H4-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of the 158P1H4
protein (or variants, homologs or analogs thereof).
[0182] III.A.) Motif-bearing Protein Embodiments
[0183] Additional illustrative embodiments of the invention
disclosed herein include 158P1H4 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within the 158P1H4 polypeptide sequence set forth in FIG. 2 or FIG.
3. Various motifs are known in the art, and a protein can be
evaluated for the presence of such motifs by a number of publicly
available Internet sites (see, e.g., URL addresses:
pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-p-
redict.html psort.ims.u-tokyo.ac.ip/; www.cbs.dtu.dk /;
www.ebi.ac.uk/interpro/scan.html;
www.expasy.ch/tools/scnpsit1.html; Epimatrix.TM. and Epimer.TM.
Brown University, www.brown.edu/Research/TB--
HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dcrt.nih.gov/.).
[0184] Motif bearing subsequences of the 158P1H4 protein are set
forth and identified in Table XIX.
[0185] Table XX sets forth several frequently occurring motifs
based on pfam searches (see URL address pfam.wustl.edu/). The
columns of Table XX list (1) motif name abbreviation, (2) percent
identity found amongst the different member of the motif family,
(3) motif name or description and (4) most common function;
location information is included if the motif is relevant for
location.
[0186] Polypeptides comprising one or more of the 158P1H4 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 158P1H4 motifs discussed above are associated with growth
dysregulation and because 158P1H4 is overexpressed in certain
cancers (See, e.g., Table I). Casein kinase II, cAMP and
camp-dependent protein kinase, and Protein Kinase C, for example,
are enzymes known to be associated with the development of the
malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):
165-174 (1998); Gaiddon et al., Endocrinology 136(10): 43314338
(1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126
(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and
O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both
glycosylation and myristoylation are protein modifications also
associated with cancer and cancer progression (see e.g. Dennis et
al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp.
Cell Res. 235(1): 145-154 (1997)). Amidation is another protein
modification also associated with cancer and cancer progression
(see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13):
169-175 (1992)).
[0187] In another embodiment, proteins of the invention comprise
one or more of the immunoreactive epitopes identified in accordance
with art-accepted methods, such as the peptides set forth in Tables
V-XVIII. CTL epitopes can be determined using specific algorithms
to identify peptides within an 158P1H4 protein that are capable of
optimally binding to specified HLA alleles (e.g., Table IV;
Epimatrix.TM. and Epimer.TM., Brown University, URL
www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatr- ix.html; and
BIMAS, URL bimas.dcrt.nih.gov/.) Moreover, processes for
identifying peptides that have sufficient binding affinity for HLA
molecules and which are correlated with being immunogenic epitopes,
are well known in the art, and are carried out without undue
experimentation. In addition, processes for identifying peptides
that are immunogenic epitopes, are well known in the art, and are
carried out without undue experimentation either in vitro or in
vivo.
[0188] Also known in the art are principles for creating analogs of
such epitopes in order to modulate immunogenicity. For example, one
begins with an epitope that bears a CTL or HTL motif (see, e.g.,
the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
The epitope is analoged by substituting out an amino acid at one of
the specified positions, and replacing it with another amino acid
specified for that position. For example, one can substitute out a
deleterious residue in favor of any other residue, such as a
preferred residue as defined in Table IV; substitute a
less-preferred residue with a preferred residue as defined in Table
IV; or substitute an originally-occurring preferred residue with
another preferred residue as defined in Table IV. Substitutions can
occur at primary anchor positions or at other positions in a
peptide; see, e.g., Table IV.
[0189] A variety of references reflect the art regarding the
identification and generation of epitopes in a protein of interest
as well as analogs thereof. See, for example, WO 9733602 to Chesnut
et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al.,
J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol.
1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4):
249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk
et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3
(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et
al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8):
3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278;
Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;
Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al.,
J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994
1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2):
79-92.
[0190] Related embodiments of the inventions include polypeptides
comprising combinations of the different motifs set forth in Table
XIX, and/or, one or more of the predicted CTL epitopes of Table V
through Table XVIII, and/or, one or more of the T cell binding
motifs known in the art. Preferred embodiments contain no
insertions, deletions or substitutions either within the motifs or
the intervening sequences of the polypeptides. In addition,
embodiments which include a number of either N-terminal and/or
C-terminal amino acid residues on either side of these motifs may
be desirable (to, for example, include a greater portion of the
polypeptide architecture in which the motif is located). Typically
the number of N-terminal and/or C-terminal amino acid residues on
either side of a motif is between about 1 to about 100 amino acid
residues, preferably 5 to about 50 amino acid residues.
[0191] 158P1H4-related proteins are embodied in many forms,
preferably in isolated form. A purified 158P1H4 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 158P1H4 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 158P1H4-related proteins include
purified 158P1 H4-related proteins and functional, soluble
158P1H4-related proteins. In one embodiment, a functional, soluble
158P1H4 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0192] The invention also provides 158P1H4 proteins comprising
biologically active fragments of the 158P1H4 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
158P1H4 protein, such as the ability to elicit the generation of
antibodies that specifically bind an epitope associated with the
158P1H4 protein; to be bound by such antibodies; to elicit the
activation of HTL or CTL; and/or, to be recognized by HTL or CTL.
158P1H4-related polypeptides that contain particularly interesting
structures can be predicted and/or identified using various
analytical techniques well known in the art, including, for
example, the methods of Chou-Fasman, Garnier-Robson,
Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf
analysis, or on the basis of immunogenicity. Fragments that contain
such structures are particularly useful in generating
subunit-specific anti-158P1H4 antibodies, or T cells or in
identifying cellular factors that bind to 158P1H4.
[0193] CTL epitopes can be determined using specific algorithms to
identify peptides within an 158P1H4 protein that are capable of
optimally binding to specified HLA alleles (e.g., by using the
SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/;
the listings in Table IV(A)-(E); Epimatrix.TM. and Epimer.TM.,
Brown University, URL (www.brown.edu/Research
WB-HIV_Lab/epimatrix/epinatrix.html); and BIMAS, URL
bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from
158P1H4 that are presented in the context of human MHC class I
molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted
(Tables V-XVIII). Specifically, the complete amino acid sequence of
the 158P1H4 protein was entered into the HLA Peptide Motif Search
algorithm found in the Bioinformatics and Molecular Analysis
Section (BIMAS) web site listed above. The HLA peptide motif search
algorithm was developed by Dr. Ken Parker based on binding of
specific peptide sequences in the groove of HLA Class I molecules,
in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6
(1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J.
Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75
(1994)). This algorithm allows location and ranking of 8-mer,
9-mer, and I 0-mer peptides from a complete protein sequence for
predicted binding to HLA-A2 as well as numerous other HLA Class I
molecules. Many HLA class I binding peptides are 8-, 9-, 10 or
11-mers. For example, for class I HLA-A2, the epitopes preferably
contain a leucine (L) or methionine (M) at position 2 and a valine
(V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J.
Immunol. 149:3580-7 (1992)). Selected results of 158P1H4 predicted
binding peptides are shown in Tables V-XVIII herein. In Tables
V-XVIII, the top 50 ranking candidates, 9-mers and 10-mers, for
each family member are shown along with their location, the amino
acid sequence of each specific peptide, and an estimated binding
score. The binding score corresponds to the estimated half time of
dissociation of complexes containing the peptide at 37.degree. C.
at pH 6.5. Peptides with the highest binding score are predicted to
be the most tightly bound to HLA Class I on the cell surface for
the greatest period of time and thus represent the best immunogenic
targets for T-cell recognition.
[0194] Actual binding of peptides to an HLA allele can be evaluated
by stabilization of HLA expression on the antigen-processing
defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8
(1997) and Peshwa et al., Prostate 36:129-38 (1998)).
Immunogenicity of specific peptides can be evaluated in vitro by
stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence
of antigen presenting cells such as dendritic cells.
[0195] It is to be appreciated that every epitope predicted by the
BIMAS site, Epimer.TM. and Epimatrix.TM. sites, or specified by the
HLA class I or class II motifs available in the art or which become
part of the art such as set forth in Table IV (or determined using
World Wide Web site URL syfpeithi.bmi-heidelberg.com/) are to be
"applied" to the 158P1H4 protein. As used in this context "applied"
means that the 158P1H4 protein is evaluated, e.g., visually or by
computer-based patterns finding methods, as appreciated by those of
skill in the relevant art. Every subsequence of the 158P1H4 of 8,
9, 10, or 11 amino acid residues that bears an HLA Class I motif,
or a subsequence of 9 or more amino acid residues that bear an HLA
Class II motif are within the scope of the invention.
[0196] III.B.) Expression of 158P1H4-related Proteins
[0197] In an embodiment described in the examples that follow,
158P1 H4 can be conveniently expressed in cells (such as 293T
cells) transfected with a commercially available expression vector
such as a CMV-driven expression vector encoding 158P1H4 with a
C-terminal 6.times.His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or
Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector
provides an IgGK secretion signal that can be used to facilitate
the production of a secreted 158P1H4 protein in transfected cells.
The secreted HIS-tagged 158P1H4 in the culture media can be
purified, e.g., using a nickel column using standard
techniques.
[0198] III.C.) Modifications of 158P1H4-related Proteins
[0199] Modifications of 158P1H4-related proteins such as covalent
modifications are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of a 158P1H4 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of the 158P1H4. Another type of covalent
modification of the 158P1H4 polypeptide included within the scope
of this invention comprises altering the native glycosylation
pattern of a protein of the invention. Another type of covalent
modification of 158P1 H4 comprises linking the 158P1H4 polypeptide
to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0200] The 158P1H4-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 158P1H4
fused to another, heterologous polypeptide or amino acid sequence.
Such a chimeric molecule can be synthesized chemically or
recombinantly. A chimeric molecule can have a protein of the
invention fused to another tumor-associated antigen or fragment
thereof. Alternatively, a protein in accordance with the invention
can comprise a fusion of fragments of the 158P1H4 sequence (amino
or nucleic acid) such that a molecule is created that is not,
through its length, directly homologous to the amino or nucleic
acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule
can comprise multiples of the same subsequence of 158P1H4. A
chimeric molecule can comprise a fusion of a 158P1H4-related
protein with a polyhistidine epitope tag, which provides an epitope
to which immobilized nickel can selectively bind, with cytokines or
with growth factors. The epitope tag is generally placed at the
amino- or carboxyl-terminus of the 158P1H4. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
158P1H4-related protein with an in immunoglobulin or a particular
region of an immunoglobulin. For a bivalent form of the chimeric
molecule (also referred to as an "immunoadhesin"), such a fusion
could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a 158P1H4 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of an IgGI
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0201] III.D.) Uses of 158P1H4-related Proteins
[0202] The proteins of the invention have a number of different
uses. As 158P1H4 is highly expressed in bladder and other cancers,
158P1H4-related proteins are used in methods that assess the status
of 158P1H4 gene products in normal versus cancerous tissues,
thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of the 158P1H4 protein are used
to assess the presence of perturbations (such as deletions,
insertions, point mutations etc.) in those regions (such as regions
containing one or more motifs). Exemplary assays utilize antibodies
or T cells targeting 158P1H4-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within the 158P1H4 polypeptide sequence in order to evaluate the
characteristics of this region in normal versus cancerous tissues
or to elicit an immune response to the epitope. Alternatively,
158P1H4-related proteins that contain the amino acid residues of
one or more of the biological motifs in the 158P1H4 protein are
used to screen for factors that interact with that region of
158P1H4.
[0203] 158P1H4 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of an 158P1H4 protein), for identifying agents or cellular
factors that bind to 158P1H4 or a particular structural domain
thereof, and in various therapeutic and diagnostic contexts,
including but not limited to diagnostic assays, cancer vaccines and
methods of preparing such vaccines.
[0204] Proteins encoded by the 158P1H4 genes, or by analogs,
homologs or fragments thereof, have a variety of uses, including
but not limited to generating antibodies and in methods for
identifying ligands and other agents and cellular constituents that
bind to an 158P1H4 gene product. Antibodies raised against an
158P1H4 protein or fragment thereof are useful in diagnostic and
prognostic assays, and imaging methodologies in the management of
human cancers characterized by expression of 158P1H4 protein, such
as those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 158P1H4-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0205] Various immunological assays useful for the detection of
158P1H4 proteins are used, including but not limited to various
types of radioimmunoassays, enzyme-linked immunosorbent assays
(ELISA), enzyme-linked immunofluorescent assays (ELIFA),
immunocytochemical methods, and the like. Antibodies can be labeled
and used as immunological imaging reagents capable of detecting
158P1H4-expressing cells (e.g., in radioscintigraphic imaging
methods). 158P1H4 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
[0206] IV.) 158P1H4 Antibodies
[0207] Another aspect of the invention provides antibodies that
bind to 158P1H4-related proteins. Preferred antibodies specifically
bind to a 158P1H4-related protein and do not bind (or bind weakly)
to peptides or proteins that are not 158P1H4-related proteins. For
example, antibodies bind 158P1H4 can bind 158P1H4-related proteins
such as the homologs or analogs thereof.
[0208] 158P1H4 antibodies of the invention are particularly useful
in bladder cancer diagnostic and prognostic assays, and imaging
methodologies. Similarly, such antibodies are useful in the
treatment, diagnosis, and/or prognosis of other cancers, to the
extent 158P1H4 is also expressed or overexpressed in these other
cancers. Moreover, intracellularly expressed antibodies (e.g.,
single chain antibodies) are therapeutically useful in treating
cancers in which the expression of 158P1H4 is involved, such as
advanced or metastatic bladder cancers.
[0209] The invention also provides various immunological assays
useful for the detection and quantification of 158P1H4 and mutant
158P1H4-related proteins. Such assays can comprise one or more
158P1H4 antibodies capable of recognizing and binding a
158P1H4-related protein, as appropriate. These assays are performed
within various immunological assay formats well known in the art,
including but not limited to various types of radioimmunoassays,
enzyme-linked immunosorbent assays (ELISA), enzyme-linked
immunofluorescent assays (ELIFA), and the like.
[0210] Immunological non-antibody assays of the invention also
comprise T cell immunogenicity assays (inhibitory or stimulatory)
as well as major histocompatibility complex (MHC) binding
assays.
[0211] In addition, immunological imaging methods capable of
detecting bladder cancer and other cancers expressing 158P1H4 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 158P1H4
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 158P1H4 expressing cancers such as
bladder cancer.
[0212] 158P1H4 antibodies are also used in methods for purifying a
158P1H4-related protein and for isolating 158P1H4 homologues and
related molecules. For example, a method of purifying a
158P1H4-related protein comprises incubating an 158P1H4 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 158P1H4-related protein under conditions that
permit the 158P1H4 antibody to bind to the 158P1H4-related protein;
washing the solid matrix to eliminate impurities; and eluting the
158P1H4-related protein from the coupled antibody. Other uses of
the 158P1H4 antibodies of the invention include generating
anti-idiotypic antibodies that mimic the 158P1H4 protein.
[0213] Various methods for the preparation of antibodies are well
known in the art. For example, antibodies can be prepared by
immunizing a suitable mammalian host using a 158P1H4-related
protein, peptide, or fragment, in isolated or immunoconjugated form
(Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane
(1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)).
In addition, fusion proteins of 158P1H4 can also be used, such as a
158P1H4 GST-fusion protein. In a particular embodiment, a GST
fusion protein comprising all or most of the amino acid sequence of
FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate
appropriate antibodies. In another embodiment, a 158P1H4-related
protein is synthesized and used as an immunogen.
[0214] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 158P1H4-related protein or
158P1H4 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0215] The amino acid sequence of 158P1H4 as shown in FIG. 2 or
FIG. 3 can be analyzed to select specific regions of the 158P1H4
protein for generating antibodies. For example, hydrophobicity and
hydrophilicity analyses of the 158P1H4 amino acid sequence are used
to identify hydrophilic regions in the 158P1H4 structure (see, e.
g., the Example entitled "Antigenicity profiles"). Regions of the
158P1H4 protein that show immunogenic structure, as well as other
regions and domains, can readily be identified using various other
methods known in the art, such as Chou-Fasman, Hopp and Woods,
Kyte-Doolittle, Janin, Bhaskaran and Ponnuswamy, Deleage and Roux,
Garnier-Robson, Eisenberg, Karplus-Schultz, or Jameson-Wolf
analysis. Thus, each region identified by any of these programs or
methods is within the scope of the present invention. Methods for
the generation of 158P1H4 antibodies are further illustrated by way
of the examples provided herein. Methods for preparing a protein or
polypeptide for use as an immunogen are well known in the art. Also
well known in the art are methods for preparing immunogenic
conjugates of a protein with a carrier, such as BSA, KLH or other
carrier protein. In some circumstances, direct conjugation using,
for example, carbodiimide reagents are used; in other instances
linking reagents such as those supplied by Pierce Chemical Co.,
Rockford, Ill., are effective. Administration of a 158P1H4
immunogen is often conducted by injection over a suitable time
period and with use of a suitable adjuvant, as is understood in the
art. During the immunization schedule, titers of antibodies can be
taken to determine adequacy of antibody formation.
[0216] 158P1H4 monoclonal antibodies can be produced by various
means well known in the art. For example, immortalized cell lines
that secrete a desired monoclonal antibody are prepared using the
standard hybridoma technology of Kohler and Milstein or
modifications that immortalize antibody-producing B cells, as is
generally known. Immortalized cell lines that secrete the desired
antibodies are screened by immunoassay in which the antigen is a
158P1H4-related protein. When the appropriate immortalized cell
culture is identified, the cells can be expanded and antibodies
produced either from in vitro cultures or from ascites fluid.
[0217] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of the 158P1H4 protein can also be produced in
the context of chimeric or complementarity determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
158P1H4 antibodies can also be produced, and are preferred for use
in therapeutic contexts. Methods for humanizing murine and other
non-human antibodies, by substituting one or more of the non-human
antibody CDRs for corresponding human antibody sequences, are well
known (see for example, Jones et al., 1986, Nature 321: 522-525;
Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al.,
1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.
Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol.
151: 2296.
[0218] Methods for producing fully human monoclonal antibodies
include phage display and transgenic methods (for review, see
Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully
human 158P1H4 monoclonal antibodies can be generated using cloning
technologies employing large human Ig gene combinatorial libraries
(i.e., phage display) (Griffiths and Hoogenboom, Building an in
vitro immune system: human antibodies from phage display libraries.
In: Protein Engineering of Antibody Molecules for Prophylactic and
Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham
Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from
combinatorial libraries. Id., pp 65-82). Fully human 158P1H4
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application W098/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued
Dec. 19, 2000; U.S. Pat. No. 6,150,584 issued Nov. 12, 2000; and,
U.S. Pat. No. 6,114,598 issued Sep. 5, 2000). This method avoids
the in vitro manipulation required with phage display technology
and efficiently produces high affinity authentic human
antibodies.
[0219] Reactivity of 158P1H4 antibodies with an 158P1H4-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 158P1H4-related proteins,
158P1H4-expressing cells or extracts thereof. A 158P1H4 antibody or
fragment thereof can be labeled with a detectable marker or
conjugated to a second molecule. Suitable detectable markers
include, but are not limited to, a radioisotope, a fluorescent
compound, a bioluminescent compound, chemiluminescent compound, a
metal chelator or an enzyme. Further, bi-specific antibodies
specific for two or more 158P1H4 epitopes are generated using
methods generally known in the art. Homodimeric antibodies can also
be generated by cross-linking techniques known in the art (e.g.,
Wolff et al., Cancer Res. 53: 2560-2565).
[0220] V.) 158P1H4 Cellular Immune Responses
[0221] The mechanism by which T cells recognize antigens has been
delineated. Efficacious peptide epitope vaccine compositions of the
invention induce a therapeutic or prophylactic immune responses in
very broad segments of the world-wide population. For an
understanding of the value and efficacy of compositions of the
invention that induce cellular immune responses, a brief review of
immunology-related technology is provided.
[0222] A complex of an HLA molecule and a peptidic antigen acts as
the ligand recognized by HLA-restricted T cells (Buus, S. et al.,
Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985;
Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989;
Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the
study of single amino acid substituted antigen analogs and the
sequencing of endogenously bound, naturally processed peptides,
critical residues that correspond to motifs required for specific
binding to HLA antigen molecules have been identified and are set
forth in Table IV (see also, e.g., Southwood, et aL, J. Immunol.
160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995;
Rammensee et al., SYFPEITHI, access via World Wide Web at URL
syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.
Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,
1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;
Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et
al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.
155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490,
1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and
Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12,
Review).
[0223] Furthermore, x-ray crystallographic analyses of HLA-peptide
complexes have revealed pockets within the peptide binding
cleft/groove of HLA molecules which accommodate, in an
allele-specific mode, residues borne by peptide ligands; these
residues in turn determine the HLA binding capacity of the peptides
in which they are present. (See, e.g., Madden, D. R. Annu. Rev.
Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont
et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994;
Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al.,
Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA
90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.
L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science
257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et
al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and
Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
[0224] Accordingly, the definition of class I and class II
allele-specific HLA binding motifs, or class I or class II
supermotifs allows identification of regions within a protein that
are correlated with binding to particular HLA antigen(s).
[0225] Thus, by a process of HLA motif identification, candidates
for epitope-based vaccines have been identified; such candidates
can be further evaluated by HLA-peptide binding assays to determine
binding affinity and/or the time period of association of the
epitope and its corresponding HLA molecule. Additional confirmatory
work can be performed to select, amongst these vaccine candidates,
epitopes with preferred characteristics in terms of population
coverage, and/or immunogenicity.
[0226] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0227] 1) Evaluation of primary T cell cultures from normal
individuals (see, e.g. Wentworth, P. A. et al., Mol. Immunol.
32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105,
1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et
al., Human Immunol. 59:1, 1998). This procedure involves the
stimulation of peripheral blood lymphocytes (PBL) from normal
subjects with a test peptide in the presence of antigen presenting
cells in vitro over a period of several weeks. T cells specific for
the peptide become activated during this time and are detected
using, e.g., a lymphokine- or .sup.51Cr-release assay involving
peptide sensitized target cells.
[0228] 2) Immunization of HLA transgenic mice (see, e.g.,
Wentworth, P. A. et aL, J. Immunol. 26:97, 1996; Wentworth, P. A.
et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J.
Immunol. 159:4753, 1997). For example, in such methods peptides in
incomplete Freund's adjuvant are administered subcutaneously to HLA
transgenic mice. Several weeks following immunization, splenocytes
are removed and cultured in vitro in the presence of test peptide
for approximately one week. Peptide-specific T cells are detected
using, e.g., a .sup.51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0229] 3) Demonstration of recall T cell responses from immune
individuals who have been either effectively vaccinated and/or from
chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp.
Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997;
Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S.
C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J.
Virol. 71:6011, 1997). Accordingly, recall responses are detected
by culturing PBL from subjects that have been exposed to the
antigen due to disease and thus have generated an immune response
"naturally", or from patients who were vaccinated against the
antigen. PBL from subjects are cultured in vitro for 1-2 weeks in
the presence of test peptide plus antigen presenting cells (APC) to
allow activation of "memory" T cells, as compared to "naive" T
cells. At the end of the culture period, T cell activity is
detected using assays including .sup.51Cr release involving
peptide-sensitized targets, T cell proliferation, or lymphokine
release.
[0230] VI.) 158P1H4 Transgenic Animals
[0231] Nucleic acids that encode a 158P1H4-related protein can also
be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. In accordance with established
techniques, cDNA encoding 158P1H4 can be used to clone genomic DNA
that encodes 158P1H4. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 158P1H4. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. No.
4,736,866 issued Apr. 12, 1988, and U.S. Pat. No. 4,870,009 issued
Sep. 26, 1989. Typically, particular cells would be targeted for
158P1H4 transgene incorporation with tissue-specific enhancers.
[0232] Transgenic animals that include a copy of a transgene
encoding 158P1H4 can be used to examine the effect of increased
expression of DNA that encodes 158P1H4. Such animals can be used as
tester animals for reagents thought to confer protection from, for
example, pathological conditions associated with its
overexpression. In accordance with this aspect of the invention, an
animal is treated with a reagent and a reduced incidence of a
pathological condition, compared to untreated animals that bear the
transgene, would indicate a potential therapeutic intervention for
the pathological condition.
[0233] Alternatively, non-human homologues of 158P1H4 can be used
to construct a 158P1H4 "knock out" animal that has a defective or
altered gene encoding 158P1H4 as a result of homologous
recombination between the endogenous gene encoding 158P1H4 and
altered genomic DNA encoding 158P1H4 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 158P1H4 can be
used to clone genomic DNA encoding 158P1H4 in accordance with
established techniques. A portion of the genomic DNA encoding
158P1H4 can be deleted or replaced with another gene, such as a
gene encoding a selectable marker that can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA
(both at the 5' and 3' ends) are included in the vector (see, e.g.,
Thomas and Capecchi, Cell, 51:503 (1987) for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced DNA has homologously recombined with the
endogenous DNA are selected (see, e.g.,, Li et al., Cell, 69:915
(1992)). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse or rat) to form aggregation chimeras (see,
e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp.
113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal, and the embryo brought to term
to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells can be identified by standard
techniques and used to breed animals in which all cells of the
animal contain the homologously recombined DNA. Knock out animals
can be characterized, for example, for their ability to defend
against certain pathological conditions or for their development of
pathological conditions due to absence of the 158P1H4
polypeptide.
[0234] VII.) Methods for the Detection of 158P1H4
[0235] Another aspect of the present invention relates to methods
for detecting 158P1H4 polynucleotides and polypeptides and
158P1H4-related proteins, as well as methods for identifying a cell
that expresses 158P1H4. The expression profile of 158P1H4 makes it
a diagnostic marker for metastasized disease. Accordingly, the
status of 158P1H4 gene products provides information useful for
predicting a variety of factors including susceptibility to
advanced stage disease, rate of progression, and/or tumor
aggressiveness. As discussed in detail herein, the status of
158P1H4 gene products in patient samples can be analyzed by a
variety protocols that are well known in the art including
immunohistochemical analysis, the variety of Northern blotting
techniques including in situ hybridization, RT-PCR analysis (for
example on laser capture micro-dissected samples), Western blot
analysis and tissue array analysis.
[0236] More particularly, the invention provides assays for the
detection of 158P1H4 polynucleotides in a biological sample, such
as urine, serum, bone, prostatic fluid, tissues, semen, cell
preparations, and the like. Detectable 158P1H4 polynucleotides
include, for example, a 158P1H4 gene or fragment thereof, 158P1H4
mRNA, alternative splice variant 158P1H4 mRNAs, and recombinant DNA
or RNA molecules that contain a 158P1H4 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 158P1H4
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0237] In one embodiment, a method for detecting an 158P1H4 mRNA in
a biological sample comprises producing cDNA from the sample by
reverse transcription using at least one primer; amplifying the
cDNA so produced using an 158P1H4 polynucleotides as sense and
antisense primers to amplify 158P1H4 cDNAs therein; and detecting
the presence of the amplified 158P1H4 cDNA. Optionally, the
sequence of the amplified 158P1H4 cDNA can be determined.
[0238] In another embodiment, a method of detecting a 158P1H4 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 158P1H4
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 158P1H4 gene. Any number of appropriate
sense and antisense probe combinations can be designed from the
nucleotide sequence provided for the 158P1H4 (FIG. 2) and used for
this purpose.
[0239] The invention also provides assays for detecting the
presence of an 158P1H4 protein in a tissue or other biological
sample such as urine, serum, semen, bone, prostate, cell
preparations, and the like. Methods for detecting a 158P1H4-related
protein are also well known and include, for example,
immunoprecipitation, immunohistochemical analysis, Western blot
analysis, molecular binding assays, ELISA, ELIFA and the like. For
example, a method of detecting the presence of a 158P1H4-related
protein in a biological sample comprises first contacting the
sample with a 158P1H4 antibody, a 158P1H4-reactive fragment
thereof, or a recombinant protein containing an antigen binding
region of a 158P1H4 antibody; and then detecting the binding of
158P1H4-related protein in the sample.
[0240] Methods for identifying a cell that expresses 158P1H4 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 158P1H4 gene comprises
detecting the presence of 158P1H4 mRNA in the cell. Methods for the
detection of particular mRNAs in cells are well known and include,
for example, hybridization assays using complementary DNA probes
(such as in situ hybridization using labeled 158P1H4 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 158P1H4, and other amplification type detection
methods, such as, for example, branched DNA, SISBA, TMA and the
like). Alternatively, an assay for identifying a cell that
expresses a 158P1H4 gene comprises detecting the presence of
158P1H4-relatedprotein in the cell or secreted by the cell. Various
methods for the detection of proteins are well known in the art and
are employed for the detection of 158P1H4-related proteins and
cells that express 158P1H4-related proteins.
[0241] 158P1H4 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 158P1H4 gene
expression. For example, 158P1H4 expression is significantly
upregulated in bladder cancer, and is expressed in cancers of the
tissues listed in Table I. Identification of a molecule or
biological agent that inhibits 158P1H4 expression or
over-expression in cancer cells is of therapeutic value. For
example, such an agent can be identified by using a screen that
quantifies 158P1H4 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0242] VIII.) Methods for Monitoring the Status of 158P1H4-related
Genes and Their Products
[0243] Oncogenesis is known to be a multistep process where
cellular growth becomes progressively dysregulated and cells
progress from a normal physiological state to precancerous and then
cancerous states (see, e.g., Alers et al., Lab Invest. 77(5):
437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)).
In this context, examining a biological sample for evidence of
dysregulated cell growth (such as aberrant 158P1H4 expression in
cancers) allows for early detection of such aberrant physiology,
before a pathologic state such as cancer has progressed to a stage
that therapeutic options are more limited and or the prognosis is
worse. In such examinations, the status of 158P1H4 in a biological
sample of interest can be compared, for example, to the status of
158P1H4 in a corresponding normal sample (e.g. a sample from that
individual or alternatively another individual that is not affected
by a pathology). An alteration in the status of 158P1H4 in the
biological sample (as compared to the normal sample) provides
evidence of dysregulated cellular growth. In addition to using a
biological sample that is not affected by a pathology as a normal
sample, one can also use a predetermined normative value such as a
predetermined normal level of mRNA expression (see, e.g., Grever et
al., J. Comp. Neurol. Dec. 9 1996;376(2):306-14 and U.S. Pat. No.
5,837,501) to compare 158P1H4 status in a sample.
[0244] The term "status" in this context is used according to its
art accepted meaning and refers to the condition or state of a gene
and its products. Typically, skilled artisans use a number of
parameters to evaluate the condition or state of a gene and its
products. These include, but are not limited to the location of
expressed gene products (including the location of 158P1H4
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 158P1H4 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 158P1H4
comprises a change in the location of 158P1H4 and/or 158P1H4
expressing cells and/or an increase in 158P1II4 mRNA and/or protein
expression.
[0245] 158P1H4 status in a sample can be analyzed by a number of
means well known in the art, including without limitation,
immunohistochemical analysis, in situ hybridization, RT-PCR
analysis on laser capture micro-dissected samples, Western blot
analysis, and tissue array analysis. Typical protocols for
evaluating the status of the 158P1H4 gene and gene products are
found, for example in Ausubel et al. eds., 1995, Current Protocols
In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern
Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the
status of 158P1H4 in a biological sample is evaluated by various
methods utilized by skilled artisans including, but not limited to
genomic Southern analysis (to examine, for example perturbations in
the 158P1H4 gene), Northern analysis and/or PCR analysis of 158P1H4
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 158P1H4 mRNAs), and, Western
and/or immunohistochemical analysis (to examine, for example
alterations in polypeptide sequences, alterations in polypeptide
localization within a sample, alterations in expression levels of
158P1H4 proteins and/or associations of 158P1H4 proteins with
polypeptide binding partners). Detectable 158P1H4 polynucleotides
include, for example, a 158P1H4 gene or fragment thereof, 158P1H4
mRNA, alternative splice variants, 158P1H4 mRNAs, and recombinant
DNA or RNA molecules containing a 158P1H4 polynucleotide.
[0246] The expression profile of 158P1 H4 makes it a diagnostic
marker for local and/or metastasized disease, and provides
information on the growth or oncogenic potential of a biological
sample. In particular, the status of 158P1H4 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 158P1H4 status and diagnosing
cancers that express 158P1H4, such as cancers of the tissues listed
in Table I. For example, because 158P1 H4 mRNA is so highly
expressed in bladder and other cancers relative to normal bladder
tissue, assays that evaluate the levels of 158P1H4 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 158P1H4 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0247] The expression status of 158P1H4 provides information
including the presence, stage and location of dysplastic,
precancerous and cancerous cells, predicting susceptibility to
various stages of disease, and/or for gauging tumor aggressiveness.
Moreover, the expression profile makes it useful as an imaging
reagent for metastasized disease. Consequently, an aspect of the
invention is directed to the various molecular prognostic and
diagnostic methods for examining the status of 158P1H4 in
biological samples such as those from individuals suffering from,
or suspected of suffering from a pathology characterized by
dysregulated cellular growth, such as cancer.
[0248] As described above, the status of 158P1H4 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 158P1H4 in a biological sample
taken from a specific location in the body can be examined by
evaluating the sample for the presence or absence of 158P1H4
expressing cells (e.g. those that express 158P1H4 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 158P1H4-expressing cells are
found in a biological sample that does not normally contain such
cells (such as a lymph node), because such alterations in the
status of 158P1H4 in a biological sample are often associated with
dysregulated cellular growth. Specifically, one indicator of
dysregulated cellular growth is the metastases of cancer cells from
an organ of origin (such as the bladder) to a different area of the
body (such as a lymph node). By example, evidence of dysregulated
cellular growth is important because occult lymph node metastases
can be detected in a substantial proportion of patients with
prostate cancer, and such metastases are associated with known
predictors of disease progression (see, e.g., Murphy et al.,
Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol.
18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt
1):474-8).
[0249] In one aspect, the invention provides methods for monitoring
158P1H4 gene products by determining the status of 158P1 H4 gene
products expressed by cells from an individual suspected of having
a disease associated with dysregulated cell growth (such as
hyperplasia or cancer) and then comparing the status so determined
to the status of 158P1H4 gene products in a corresponding normal
sample. The presence of aberrant 158P1H4 gene products in the test
sample relative to the normal sample provides an indication of the
presence of dysregulated cell growth within the cells of the
individual.
[0250] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 158P1H4 mRNA or protein
expression in a test cell or tissue sample relative to expression
levels in the corresponding normal cell or tissue. The presence of
158P1H4 mRNA can, for example, be evaluated in tissue samples
including but not limited to those listed in Table I. The presence
of significant 158P1H4 expression in any of these tissues is useful
to indicate the emergence, presence and/or severity of a cancer,
since the corresponding normal tissues do not express 158P1H4 mRNA
or express it at lower levels.
[0251] In a related embodiment, 158P1H4 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 158P1H4 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 158P1H4 expressed in a corresponding
normal sample. In one embodiment, the presence of 158P1H4 protein
is evaluated, for example, using immunohistochemicalmethods.
158P1H4antibodies or binding partners capable of detecting 158P1H4
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0252] In a further embodiment, one can evaluate the status of
158P1H4 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules. These perturbations can include insertions, deletions,
substitutions and the like. Such evaluations are useful because
perturbations in the nucleotide and amino acid sequences are
observed in a large number of proteins associated with a growth
dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.
Pathol. 26(8):369-378). For example, a mutation in the sequence of
158P1H4 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 158P1H4 indicates a potential loss of function or
increase in tumor growth.
[0253] A wide variety of assays for observing perturbations in
nucleotide and amino acid sequences are well known in the art. For
example, the size and structure of nucleic acid or amino acid
sequences of 158P1H4 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. No. 5,382,510 issued Sep. 7, 1999, and U.S. Pat.
No. 5,952,170 issued Jan. 17, 1995).
[0254] Additionally, one can examine the methylation status of the
158P1H4 gene in a biological sample. Aberrant demethylation and/or
hypermethylation of CpG islands in gene 5' regulatory regions
frequently occurs in immortalized and transformed cells, and can
result in altered expression of various genes. For example,
promoter hypermethylation of the DBCCR1, PAX6 and APC genes have
been detected in bladder cancers leading to aberrant expression of
the genes (Esteller et al., Cancer Res 2001; 61:3225-3229). A
variety of assays for examining methylation status of a gene are
well known in the art. For example, one can utilize, in Southern
hybridization approaches, methylation-sensitive restriction enzymes
which cannot cleave sequences that contain methylated CpG sites to
assess the methylation status of CpG islands. In addition, MSP
(methylation specific PCR) can rapidly profile the methylation
status of all the CpG sites present in a CpG island of a given
gene. This procedure involves initial modification of DNA by sodium
bisulfite (which will convert all unmethylated cytosines to uracil)
followed by amplification using primers specific for methylated
versus unmethylated DNA. Protocols involving methylation
interference can also be found for example in Current Protocols In
Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds.,
1995.
[0255] Gene amplification is an additional method for assessing the
status of 158P1H4. Gene amplification is measured in a sample
directly, for example, by conventional Southern blotting or
Northern blotting to quantitate the transcription of mRNA (Thomas,
1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies are employed that recognize specific duplexes, including
DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or
DNA-protein duplexes. The antibodies in turn are labeled and the
assay carried out where the duplex is bound to a surface, so that
upon the formation of duplex on the surface, the presence of
antibody bound to the duplex can be detected.
[0256] Biopsied tissue or peripheral blood can be conveniently
assayed for the presence of cancer cells using for example,
Northern, dot blot or RT-PCR analysis to detect 158P1H4 expression.
The presence of RT-PCR amplifiable 158P1H4 mRNA provides an
indication of the presence of cancer. RT-PCR assays are well known
in the art RT-PCR detection assays for tumor cells in peripheral
blood are currently being evaluated for use in the diagnosis and
management of a number of human solid tumors.
[0257] A further aspect of the invention is an assessment of the
susceptibility that an individual has for developing cancer. In one
embodiment, a method for predicting susceptibility to cancer
comprises detecting 158P1H4 mRNA or 158P1H4 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 158P1H4 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 158P1H4
in bladder or other tissue is examined, with the presence of
158P1H4 in the sample providing an indication of bladder cancer
susceptibility (or the emergence or existence of a bladder tumor).
Similarly, one can evaluate the integrity 158P1H4 nucleotide and
amino acid sequences in a biological sample, in order to identify
perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The presence of
one or more perturbations in 158P1H4 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0258] The invention also comprises methods for gauging tumor
aggressiveness. In one embodiment, a method for gauging
aggressiveness of a tumor comprises determining the level of
158P1H4 mRNA or 158P1H4 protein expressed by tumor cells, comparing
the level so determined to the level of 158P1H4 mRNA or 158P1H4
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 158P1H4 mRNA or 158P1H4 protein expression in the tumor
sample relative to the normal sample indicates the degree of
aggressiveness. In a specific embodiment, aggressiveness of a tumor
is evaluated by determining the extent to which 158P1H4 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 158P1H4 nucleotide and amino acid
sequences in a biological sample, in order to identify
perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The presence of
one or more perturbations indicates more aggressive tumors.
[0259] Another embodiment of the invention is directed to methods
for observing the progression of a malignancy in an individual over
time. In one embodiment, methods for observing the progression of a
malignancy in an individual over time comprise determining the
level of 158P1H4 mRNA or 158P1H4 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 158P1H4 mRNA or 158P1H4 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 158P1H4 mRNA or 158P1H4 protein expression in
the tumor sample over time provides information on the progression
of the cancer. In a specific embodiment, the progression of a
cancer is evaluated by determining 158P1H4 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
158P1H4 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules such as insertions, deletions, substitutions and the
like, where the presence of one or more perturbations indicates a
progression of the cancer.
[0260] The above diagnostic approaches can be combined with any one
of a wide variety of prognostic and diagnostic protocols known in
the art. For example, another embodiment of the invention is
directed to methods for observing a coincidence between the
expression of 158P1H4 gene and 158P1H4 gene products (or
perturbations in 158P1H4 gene and 158P1H4 gene products) and a
factor that is associated with malignancy, as a means for
diagnosing and prognosticating the status of a tissue sample. A
wide variety of factors associated with malignancy can be utilized,
such as the expression of genes associated with malignancy (e.g.
PSCA, H-ras and p53 expression etc.) as well as gross cytological
observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.
6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et
al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am J.
Surg. Pathol. 23(8):918-24). Methods for observing a coincidence
between the expression of 158P1H4 gene and 158P1H4 gene products
(or perturbations in 158P1H4 gene and 158P1H4 gene products) and
another factor that is associated with malignancy are useful, for
example, because the presence of a set of specific factors that
coincide with disease provides information crucial for diagnosing
and prognosticating the status of a tissue sample.
[0261] In one embodiment, methods for observing a coincidence
between the expression of 158P1H4 gene and 158P1H4 gene products
(or perturbations in 158P1H4 gene and 158P1H4 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 158P1H4 mRNA or protein in a tissue sample,
detecting the overexpression of BLCA-4A mRNA or protein in a tissue
sample (or PSCA expression), and observing a coincidence of 158P1H4
mRNA or protein and BLCA4 mRNA or protein overexpression (or PSCA
expression) (Amara et al., 2001, Cancer Res 61:4660-4665; Konety et
al., Clin Cancer Res, 2000, 6(7):2618-2625). In a specific
embodiment, the expression of 158P1H4 and BLCA4 mRNA in bladder
tissue is examined, where the coincidence of 158P1H4 and BLCA4 mRNA
overexpression in the sample indicates the existence of bladder
cancer, bladder cancer susceptibility or the emergence or status of
a bladder tumor.
[0262] Methods for detecting and quantifying the expression of
158P1H4 mRNA or protein are described herein, and standard nucleic
acid and protein detection and quantification technologies are well
known in the art. Standard methods for the detection and
quantification of 158P1H4 mRNA include in situ hybridization using
labeled 158P1H4 riboprobes, Northern blot and related techniques
using 158P1H4 polynucleotide probes, RT-PCR analysis using primers
specific for 158P1H4, and other amplification type detection
methods, such as, for example, branched DNA, SISBA, TMA and the
like. In a specific embodiment, semi-quantitative RT-PCR is used to
detect and quantify 158P1H4 mRNA expression. Any number of primers
capable of amplifying 158P1H4 can be used for this purpose,
including but not limited to the various primer sets specifically
described herein. In a specific embodiment, polyclonal or
monoclonal antibodies specifically reactive with the wild-type
158P1H4 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0263] IX.) Identification of Molecules that Interact with
158P1H4
[0264] The 158P1H4 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 158P1H4, as well as
pathways activated by 158P1H4 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. No. 5,955,280 issued Sep. 21, 1999,
U.S. Pat. No. 5,925,523 issued Jul. 20, 1999, U.S. Pat. No.
5,846,722 issued Dec. 8, 1998 and U.S. Pat. No. 6,004,746 issued
Dec. 21, 1999. Algorithms are also available in the art for
genome-based predictions of protein function (see, e.g., Marcotte,
et al., Nature 402: Nov. 4, 1999, 83-86).
[0265] Alternatively one can screen peptide libraries to identify
molecules that interact with 158P1H4 protein sequences. In such
methods, peptides that bind to 158P1H4 are identified by screening
libraries that encode a random or controlled collection of amino
acids. Peptides encoded by the libraries are expressed as fusion
proteins of bacteriophage coat proteins, the bacteriophage
particles are then screened against the 158P1H4 protein.
[0266] Accordingly, peptides having a wide variety of uses, such as
therapeutic, prognostic or diagnostic reagents, are thus identified
without any prior information on the structure of the expected
ligand or receptor molecule. Typical peptide libraries and
screening methods that can be used to identify molecules that
interact with 158P1H4 protein sequences are disclosed for example
in U.S. Pat. No. 5,723,286 issued Mar. 3, 1998 and U.S. Pat. No.
5,733,731 issued Mar. 31, 1998.
[0267] Alternatively, cell lines that express 158P1H4 are used to
identify protein-protein interactions mediated by 158P1H4. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B J, et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 158P1H4 protein can be immunoprecipitated from
158P1H4-expressing cell lines using anti-158P1H4 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 158P1H4 and a His-tag
(vectors mentioned above). The immunoprecipitated complex can be
examined for protein association by procedures such as Western
blotting, .sup.35S-methionine labeling of proteins, protein
microsequencing, silver staining and two-dimensional gel
electrophoresis.
[0268] Small molecules and ligands that interact with 158P1H4 can
be identified through related embodiments of such screening assays.
For example, small molecules can be identified that interfere with
protein function, including molecules that interfere with 158P1H4's
ability to mediate phosphorylation and de-phosphorylation,
interaction with DNA or RNA molecules as an indication of
regulation of cell cycles, second messenger signaling or
tumorigenesis. Similarly, small molecules that modulate 158P1H4
related ion channel, protein pump, or cell communication functions
are identified and used to treat patients that have a cancer that
expresses 158P1H4 (see, e.g., Hille, B., Ionic Channels of
Excitable Membranes 2.sup.nd Ed., Sinauer Assoc., Sunderland,
Mass., 1992). Moreover, ligands that regulate 158P1H4 function can
be identified based on their ability to bind 158P1H4 and activate a
reporter construct. Typical methods are discussed for example in
U.S. Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods
for forming hybrid ligands in which at least one ligand is a small
molecule. In an illustrative embodiment, cells engineered to
express a fusion protein of 158P1H4 and a DNA-binding protein are
used to co-express a fusion protein of a hybrid ligand/small
molecule and a cDNA library transcriptional activator protein. The
cells further contain a reporter gene, the expression of which is
conditioned on the proximity of the first and second fusion
proteins to each other, an event that occurs only if the hybrid
ligand binds to target sites on both hybrid proteins. Those cells
that express the reporter gene are selected and the unknown small
molecule or the unknown ligand is identified. This method provides
a means of identifying modulators which activate or inhibit
158P1H4.
[0269] An embodiment of this invention comprises a method of
screening for a molecule that interacts with an 158P1H4 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with the 158P1H4 amino acid
sequence, allowing the population of molecules and the 158P1H4
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 158P1H4 amino acid sequence, and then separating molecules
that do not interact with the 158P1H4 amino acid sequence from
molecules that do. In a specific embodiment, the method further
comprises purifying, characterizing and identifying a molecule that
interacts with the 158P1H4 amino acid sequence. The identified
molecule can be used to modulate a function performed by 158P1H4.
In a preferred embodiment, the 158P1H4 amino acid sequence is
contacted with a library of peptides.
[0270] X.) Therapeutic Methods and Compositions
[0271] The identification of 158P1H4 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in bladder and other cancers, opens a number of
therapeutic approaches to the treatment of such cancers. As
contemplated herein, 158P1H4 functions as a transcription factor
involved in activating tumor-promoting genes or repressing genes
that block tumorigenesis.
[0272] Accordingly, therapeutic approaches that inhibit the
activity of the 158P1H4 protein are useful for patients suffering
from a cancer that expresses 158P1H4. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of the 158P1H4
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of the 158P1 H4 gene or translation of 158P1H4
mRNA.
[0273] X.A.) Anti-Cancer Vaccines
[0274] The invention provides cancer vaccines comprising a
158P1H4-related protein or 158P1H4-related nucleic acid. In view of
the expression of 158P1H4, cancer vaccines prevent and/or treat
158P1H4-expressing cancers with minimal or no effects on non-target
tissues. The use of a tumor antigen in a vaccine that generates
humoral and/or cell-mediated immune responses as anti-cancer
therapy is well known in the art (see, e.g., Hodge et al., 1995,
Int J. Cancer 63:231-237; Fong et al., 1997, J. Inmunol.
159:3113-3117).
[0275] Such methods can be readily practiced by employing a
158P1H4-related protein, or a 158P1H4-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 158P1H4 immunogen (which typically comprises a
number of antibody or T cell epitopes). Skilled artisans understand
that a wide variety of vaccine systems for delivery of
immunoreactive epitopes are known in the art (see, e.g., Heryln et
al., Ann Med February 1999 31(1):66-78; Maruyama et al., Cancer
Immunol Immunother June 2000 49(3):123-32) Briefly, such methods of
generating an immune response (e.g. humoral and/or cell-mediated)
in a mammal, comprise the steps of: exposing the mammal's immune
system to an immunoreactive epitope (e.g. an epitope present in the
158P1H4 protein shown in SEQ ID NO: 703 or analog or homolog
thereof) so that the mammal generates an immune response that is
specific for that epitope (e.g. generates antibodies that
specifically recognize that epitope). In a preferred method, the
158P1H4 immunogen contains a biological motif, see e.g., Tables
V-XVIII, or a peptide of a size range from 158P1H4 indicated in
FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13.
[0276] The entire 158P1H4 protein, immunogenic regions or epitopes
thereof can be combined and delivered by various means. Such
vaccine compositions can include, for example, lipopeptides (e.g.,
Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide
compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG")
microspheres (see, e.g., Eldridge, et al., Molec. Immunol.
28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et
al., Vaccine 13:675-681, 1995), peptide compositions contained in
immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al.,
Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243,
1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J.
P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P.,
J. Immunol. Methods 196:17-32, 1996), peptides formulated as
multivalent peptides; peptides for use in ballistic delivery
systems, typically crystallized peptides, viral delivery vectors
(Perkus, M. E. et al., In: Concepts in vaccine development,
Kaufmarn, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al.,
Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;
Kieny, M.-P. et al., AIDS BiolTechnology 4:790, 1986; Top, F. H. et
al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology
175:535, 1990), particles of viral or synthetic origin (e.g.,
Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J.
H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al.,
Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,
and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et
al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J.
Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996),
or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science
259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G.,
Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine
development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B.,
and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and
Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted
delivery technologies, also known as receptor mediated targeting,
such as those of Avant Immunotherapeutics, Inc. (Needham,
Massachusetts) may also be used.
[0277] In patients with 158P1H4-associated cancer, the vaccine
compositions of the invention can also be used in conjunction with
other treatments used for cancer, e.g., surgery, chemotherapy, drug
therapies, radiation therapies, etc. including use in combination
with immune adjuvants such as IL-2, IL-12, GM-CSF, and the
like.
[0278] Cellular Vaccines
[0279] CTL epitopes can be determined using specific algorithms to
identify peptides within 158P1H4 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University (URL
www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.htm- l); and,
BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL
syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, the
158P1H4 immunogen contains one or more amino acid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables V-XVIII or a peptide of 8, 9, 10 or 11
amino acids specified by an HLA Class I motif/supermotif (e.g.,
Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at
least 9 amino acids that comprises an HLA Class II motif/supermotif
(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art,
the HLA Class I binding groove is essentially closed ended so that
peptides of only a particular size range can fit into the groove
and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11
amino acids long. In contrast, the HLA Class II binding groove is
essentially open ended; therefore a peptide of about 9 or more
amino acids can be bound by an HLA Class II molecule. Due to the
binding groove differences between HLA Class I and II, HLA Class I
motifs are length specific, i.e., position two of a Class I motif
is the second amino acid in an amino to carboxyl direction of the
peptide. The amino acid positions in a Class II motif are relative
only to each other, not the overall peptide, i.e., additional amino
acids can be attached to the amino and/or carboxyl termini of a
motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino
acids long, or longer than 25 amino acids.
[0280] Antibody-based Vaccines
[0281] A wide variety of methods for generating an immune response
in a mammal are known in the art (for example as the first step in
the generation of hybridomas). Methods of generating an immune
response in a mammal comprise exposing the mammal's immune system
to an immunogenic epitope on a protein (e.g. the 158P1H4 protein)
so that an immune response is generated. A typical embodiment
consists of a method for generating an immune response to 158P I H4
in a host, by contacting the host with a sufficient amount of at
least one 158P1H4 B cell or cytotoxic T-cell epitope or analog
thereof; and at least one periodic interval thereafter
re-contacting the host with the 158P1H4 B cell or cytotoxic T-cell
epitope or analog thereof. A specific embodiment consists of a
method of generating an immune response against a 158P1H4-related
protein or a man-made multiepitopic peptide comprising:
administering 158P1H4 immunogen (e.g. the 158P1H4 protein or a
peptide fragment thereof, an 158P1H4 fusion protein or analog etc.)
in a vaccine preparation to a human or another mammal. Typically,
such vaccine preparations further contain a suitable adjuvant (see,
e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such
as a PADRE.TM. peptide (Epimmune Inc., San Diego, Calif.; see,
e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;
Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al.,
Immunol. Res. 1998 18(2): 79-92). An alternative method comprises
generating an immune response in an individual against a 158P1H4
immunogen by: administering in vivo to muscle or skin of the
individual's body a DNA molecule that comprises a DNA sequence that
encodes an 158P1H4 immunogen, the DNA sequence operatively linked
to regulatory sequences which control the expression of the DNA
sequence; wherein the DNA molecule is taken up by cells, the DNA
sequence is expressed in the cells and an immune response is
generated against the immunogen (see, e.g., U.S. Pat. No.
5,962,428). Optionally a genetic vaccine facilitator such as
anionic lipids; saponins; lectins; estrogenic compounds;
hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also
administered.
[0282] Nucleic Acid Vaccines
[0283] Vaccine compositions of the invention include nucleic
acid-mediated modalities. DNA or RNA that encode protein(s) of the
invention can be administered to a patient. Genetic immunization
methods can be employed to generate prophylactic or therapeutic
humoral and cellular immune responses directed against cancer cells
expressing 158P1H4. Constructs comprising DNA encoding a
158P1H4-related protein/immunogen and appropriate regulatory
sequences can be injected directly into muscle or skin of an
individual, such that the cells of the muscle or skin take-up the
construct and express the encoded 158P1H4 protein/immunogen.
Alternatively, a vaccine comprises a 158P1H4-related protein.
Expression of the 158P1H4-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear 158P1H4 protein. Various
prophylactic and therapeutic genetic immunization techniques known
in the art can be used (for review, see information and references
published at Internet address www.genweb.com). Nucleic acid-based
delivery is described, for instance, in Wolff et. al., Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples
of DNA-based delivery technologies include "naked DNA", facilitated
(bupivicaine, polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0284] For therapeutic or prophylactic immunization purposes,
proteins of the invention can be expressed via viral or bacterial
vectors. Various viral gene delivery systems that can be used in
the practice of the invention include, but are not limited to,
vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovims,
adeno-associated virus, lentivirus, and sindbis virus (see, e.g.,
Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J.
Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems
can also be employed by introducing naked DNA encoding a
158P1H4-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0285] Vaccinia virus is used, for example, as a vector to express
nucleotide sequences that encode the peptides of the invention.
Upon introduction into a host, the recombinant vaccinia virus
expresses the protein immunogenic peptide, and thereby elicits a
host immune response. Vaccinia vectors and methods useful in
immunization protocols are described in, e.g., U.S. Pat. No.
4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG
vectors are described in Stover et al., Nature 351:456-460 (1991).
A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention,
e.g. adeno and adeno-associated virus vectors, retroviral vectors,
Salmonella typhi vectors, detoxified anthrax toxin vectors, and the
like, will be apparent to those skilled in the art from the
description herein.
[0286] Thus, gene delivery systems are used to deliver a
158P1H4-related nucleic acid molecule. In one embodiment, the
full-length human 158P1H4 cDNA is employed. In another embodiment,
158P1H4 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0287] Ex Vivo Vaccines
[0288] Various ex vivo strategies can also be employed to generate
an immune response. One approach involves the use of antigen
presenting cells (APCs) such as dendritic cells (DC) to present
158P1H4 antigen to a patient's immune system. Dendritic cells
express MHC class I and II molecules, B7 co-stimulator, and IL-12,
and are thus highly specialized antigen presenting cells. In
bladder cancer, autologous dendritic cells pulsed with peptides of
the MAGE-3 antigen are being used in a Phase I clinical trial to
stimulate bladder cancer patients' immune systems (Nishiyama et
al., 2001, Clin Cancer Res, 7(l):23-31). Thus, dendritic cells can
be used to present 158P1H4 peptides to T cells in the context of
MHC class I or II molecules. In one embodiment, autologous
dendritic cells are pulsed with 158P1H4 peptides capable of binding
to MHC class I and/or class II molecules. In another embodiment,
dendritic cells are pulsed with the complete 158P1H4 protein. Yet
another embodiment involves engineering the overexpression of the
158P1H4 gene in dendritic cells using various implementing vectors
known in the art, such as adenovirus (Arthur et al., 1997, Cancer
Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer
Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA
transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or
tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.
186:1177-1182). Cells that express 158P1H4 can also be engineered
to express immune modulators, such as GM-CSF, and used as
immunizing agents.
[0289] X.B.) 158P1H4 as a Target for Antibody-based Therapy
[0290] 158P1H4 is an attractive target for antibody-based
therapeutic strategies. A number of antibody strategies are known
in the art for targeting both extracellular and intracellular
molecules (see, e.g., complement and ADCC mediated killing as well
as the use of intrabodies). Because 158P1H4 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 158P1H4-immunoreactive compositions are
prepared that exhibit excellent sensitivity without toxic,
non-specific and/or non-target effects caused by binding of the
immunoreactive composition to non-target organs and tissues.
Antibodies specifically reactive with domains of 158P1H4 are useful
to treat 158P1H4-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0291] 158P1H4 antibodies can be introduced into a patient such
that the antibody binds to 158P1H4 and modulates a function, such
as an interaction with a binding partner, and consequently mediates
destruction of the tumor cells and/or inhibits the growth of the
tumor cells. Mechanisms by which such antibodies exert a
therapeutic effect can include complement-mediated cytolysis,
antibody-dependent cellular cytotoxicity, modulation of the
physiological function of 158P1H4, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0292] Those skilled in the art understand that antibodies can be
used to specifically target and bind immunogenic molecules such as
an immunogenic region of the 158P1H4 sequence shown in FIG. 2 or
FIG. 3. In addition, skilled artisans understand that it is routine
to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et
al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or
therapeutic agents are delivered directly to cells, such as by
conjugating them to antibodies specific for a molecule expressed by
that cell (e.g. 158P1H4), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0293] A wide variety of compositions and methods for using
antibody-cytotoxic agent conjugates to kill cells are known in the
art. In the context of cancers, typical methods entail
administering to an animal having a tumor a biologically effective
amount of a conjugate comprising a selected cytotoxic and/or
therapeutic agent linked to a targeting agent (e.g. an anti-158P1H4
antibody) that binds to a marker (e.g. 158P1H4) expressed,
accessible to binding or localized on the cell surfaces. A typical
embodiment is a method of delivering a cytotoxic and/or therapeutic
agent to a cell expressing 158P1H4, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
158P1H4 epitope, and, exposing the cell to the antibody-agent
conjugate. Another illustrative embodiment is a method of treating
an individual suspected of suffering from metastasized cancer,
comprising a step of administering parenterally to said individual
a pharmaceutical composition comprising a therapeutically effective
amount of an antibody conjugated to a cytotoxic and/or therapeutic
agent.
[0294] Cancer immunotherapy using anti-158P1H4 antibodies can be
done in accordance with various approaches that have been
successfully employed in the treatment of other types of cancer,
including but not limited to colon cancer (Arlen et al., 1998,
Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al.,
1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood
90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.
52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.
Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et
al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al.,
1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.
55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.
Immunol. 11: 117-127). Some therapeutic approaches involve
conjugation of naked antibody to a toxin, such as the conjugation
of Y.sup.91 or I.sup.131 to anti-CD20 antibodies (e.g.,
Zevalin.TM., IDEC Pharmaceuticals Corp. or Bexxar.TM., Coulter
Pharmaceuticals), while others involve co-administration of
antibodies and other therapeutic agents, such as Herceptin.TM.
(trastuzumab) with paclitaxel (Genentech, Inc.). To treat bladder
cancer, for example, 158P1H4 antibodies can be administered in
conjunction with radiation, chemotherapy or hormone ablation.
[0295] Although 158P1H4 antibody therapy is useful for all stages
of cancer, antibody therapy can be particularly appropriate in
advanced or metastatic cancers. Treatment with the antibody therapy
of the invention is indicated for patients who have received one or
more rounds of chemotherapy. Alternatively, antibody therapy of the
invention is combined with a chemotherapeutic or radiation regimen
for patients who have not received chemotherapeutic treatment.
Additionally, antibody therapy can enable the use of reduced
dosages of concomitant chemotherapy, particularly for patients who
do not tolerate the toxicity of the chemotherapeutic agent very
well.
[0296] Cancer patients can be evaluated for the presence and level
of 158P1H4 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 158P1H4 imaging, or other
techniques that reliably indicate the presence and degree of
158P1H4 expression. Immunohistochemical analysis of tumor biopsies
or surgical specimens is preferred for this purpose. Methods for
immunohistochemical analysis of tumor tissues are well known in the
art.
[0297] Anti-158P1H4 monoclonal antibodies that treat bladder and
other cancers include those that initiate a potent immune response
against the tumor or those that are directly cytotoxic. In this
regard, anti-158P1H4 monoclonal antibodies (mAbs) can elicit tumor
cell lysis by either complement-mediated or antibody-dependent cell
cytotoxicity (ADCC) mechanisms, both of which require an intact Fc
portion of the immunoglobulin molecule for interaction with
effector cell Fc receptor sites on complement proteins. In
addition, anti-158P1H4 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 158P1H4.
Mechanisms by which directly cytotoxic mAbs act include: inhibition
of cell growth, modulation of cellular differentiation, modulation
of tumor angiogenesis factor profiles, and the induction of
apoptosis. The mechanism(s) by which a particular anti-158P1H4 mAb
exerts an anti-tumor effect is evaluated using any number of in
vitro assays that evaluate cell death such as ADCC, ADMMC,
complement-mediated cell lysis, and so forth, as is generally known
in the art.
[0298] In some patients, the use of murine or other non-human
monoclonal antibodies, or human/mouse chimeric mAbs can induce
moderate to strong immune responses against the non-human antibody.
This can result in clearance of the antibody from circulation and
reduced efficacy. In the most severe cases, such an immune response
can lead to the extensive formation of immune complexes which,
potentially, can cause renal failure. Accordingly, preferred
monoclonal antibodies used in the therapeutic methods of the
invention are those that are either fully human or humanized and
that bind specifically to the target 158P1H4 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0299] Therapeutic methods of the invention contemplate the
administration of single anti-158P1H4 mAbs as well as combinations,
or cocktails, of different mAbs. Such mAb cocktails can have
certain advantages inasmuch as they contain mAbs that target
different epitopes, exploit different effector mechanisms or
combine directly cytotoxic mAbs with mAbs that rely on immune
effector functionality. Such mAbs in combination can exhibit
synergistic therapeutic effects. In addition, anti-158P1H4 mAbs can
be administered concomitantly with other therapeutic modalities,
including but not limited to various chemotherapeutic agents,
androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery
or radiation. The anti-158P1H4 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0300] Anti-158P1H4 antibody formulations are administered via any
route capable of delivering the antibodies to a tumor cell. Routes
of administration include, but are not limited to, intravenous,
intraperitoneal, intramuscular, intratumor, intradermal, and the
like. Treatment generally involves repeated administration of the
anti-158P1H4 antibody preparation, via an acceptable route of
administration such as intravenous injection (IV), typically at a
dose in the range of about 0.1 to about 10 mg/kg body weight. In
general, doses in the range of 10-500 mg mAb per week are effective
and well tolerated.
[0301] Based on clinical experience with the Herceptin mAb in the
treatment of metastatic breast cancer, an initial loading dose of
approximately 4 mg/kg patient body weight IV, followed by weekly
doses of about 2 mg/kg IV of the anti- 158P1 H4 mAb preparation
represents an acceptable dosing regimen. Preferably, the initial
loading dose is administered as a 90 minute or longer infusion. The
periodic maintenance dose is administered as a 30 minute or longer
infusion, provided the initial dose was well tolerated. As
appreciated by those of skill in the art, various factors can
influence the ideal dose regimen in a particular case. Such factors
include, for example, the binding affinity and half life of the Ab
or mAbs used, the degree of 158P1H4 expression in the patient, the
extent of circulating shed 158P1H4 antigen, the desired
steady-state antibody concentration level, frequency of treatment,
and the influence of chemotherapeutic or other agents used in
combination with the treatment method of the invention, as well as
the health status of a particular patient.
[0302] Optionally, patients should be evaluated for the levels of
158P1H4 in a given sample (e.g. the levels of circulating 158P1H4
antigen and/or 158P1H4 expressing cells) in order to assist in the
determination of the most effective dosing regimen, etc. Such
evaluations are also used for monitoring purposes throughout
therapy, and are useful to gauge therapeutic success in combination
with the evaluation of other parameters (for example, urine
cytology and/or ImmunoCyt levels in bladder cancer therapy, or by
analogy, serum PSA levels in prostate cancer therapy).
[0303] Anti-idiotypic anti-158P1H4 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 158P1H4-related protein. In particular, the
generation of anti-idiotypic antibodies is well known in the art;
this methodology can readily be adapted to generate anti-idiotypic
anti-158P1H4 antibodies that mimic an epitope on a 158P1H4-related
protein (see, for example, Wagner et al., 1997, Hybridoma 16:
33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et
al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an
anti-idiotypic antibody can be used in cancer vaccine
strategies.
[0304] X.C.) 158P1H4 as a Target for Cellular Immune Responses
[0305] Vaccines and methods of preparing vaccines that contain an
immunogenically effective amount of one or more HLA-binding
peptides as described herein are further embodiments of the
invention. Furthermore, vaccines in accordance with the invention
encompass compositions of one or more of the claimed peptides. A
peptide can be present in a vaccine individually. Alternatively,
the peptide can exist as a homopolymer comprising multiple copies
of the same peptide, or as a heteropolymer of various peptides.
Polymers have the advantage of increased immunological reaction
and, where different peptide epitopes are used to make up the
polymer, the additional ability to induce antibodies and/or CTLs
that react with different antigenic determinants of the pathogenic
organism or tumor-related peptide targeted for an immune response.
The composition can be a naturally occurring region of an antigen
or can be prepared, e.g., recombinantly or by chemical
synthesis.
[0306] Carriers that can be used with vaccines of the invention are
well known in the art, and include, e.g., thyroglobulin, albumins
such as human serum albumin, tetanus toxoid, polyamino acids such
as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B
virus core protein, and the like. The vaccines can contain a
physiologically tolerable (i.e., acceptable) diluent such as water,
or saline, preferably phosphate buffered saline. The vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, as
disclosed herein, CTL responses can be primed by conjugating
peptides of the invention to lipids, such as
tripalmitoyl-S-glycerylcysteinlyseryl- serine (P.sub.3CSS).
Moreover, an adjuvant such as a synthetic
cytosine-phosphorothiolated-guanine-containi- ng (CpG)
oligonucleotides has been found to increase CTL responses 10- to
100-fold. (see, e.g. Davila and Celis J. Immunol. 165:539-547
(2000))
[0307] Upon immunization with a peptide composition in accordance
with the invention, via injection, aerosol, oral, transdermal,
transmucosal, intrapleural, intrathecal, or other suitable routes,
the immune system of the host responds to the vaccine by producing
large amounts of CTLs and/or HTLs specific for the desired antigen.
Consequently, the host becomes at least partially immune to later
development of cells that express or overexpress 158P1H4 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0308] In some embodiments, it may be desirable to combine the
class I peptide components with components that induce or
facilitate neutralizing antibody and or helper T cell responses
directed to the target antigen. A preferred embodiment of such a
composition comprises class I and class II epitopes in accordance
with the invention. An alternative embodiment of such a composition
comprises a class I and/or class II epitope in accordance with the
invention, along with a cross reactive HTL epitope such as
PADRE.TM. (Epimmune, San Diego, Calif.) molecule (described e.g.,
in U.S. Pat. No. 5,736,142).
[0309] A vaccine of the invention can also include
antigen-presenting cells (APC), such as dendritic cells (DC), as a
vehicle to present peptides of the invention. Vaccine compositions
can be created in vitro, following dendritic cell mobilization and
harvesting, whereby loading of dendritic cells occurs in vitro. For
example, dendritic cells are transfected, e.g., with a minigene in
accordance with the invention, or are pulsed with peptides. The
dendritic cell can then be administered to a patient to elicit
immune responses in vivo. Vaccine compositions, either DNA- or
peptide-based, can also be administered in vivo in combination with
dendritic cell mobilization whereby loading of dendritic cells
occurs in vivo.
[0310] Preferably, the following principles are utilized when
selecting an array of epitopes for inclusion in a polyepitopic
composition for use in a vaccine, or for selecting discrete
epitopes to be included in a vaccine and/or to be encoded by
nucleic acids such as a minigene. It is preferred that each of the
following principles be balanced in order to make the selection.
The multiple epitopes to be incorporated in a given vaccine
composition may be, but need not be, contiguous in sequence in the
native antigen from which the epitopes are derived.
[0311] 1.) Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with
tumor clearance. For HLA Class I this includes 3-4 epitopes that
come from at least one tumor associated antigen (TAA). For HLA
Class II a similar rationale is employed; again 3-4 epitopes are
selected from at least one TAA (see, e.g., Rosenberg et al, Science
278:1447-1450). Epitopes from one TAA may be used in combination
with epitopes from one or more additional TAAs to produce a vaccine
that targets tumors with varying expression patterns of
frequently-expressed TAAs.
[0312] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC.sub.50 of 500 nM or less, often 200 nM or less; and
for Class II an IC.sub.50 of 1000 nM or less.
[0313] 3.) Sufficient supermotif bearing-peptides, or a sufficient
array of allele-specific motif-bearing peptides, are selected to
give broad population coverage. For example, it is preferable to
have at least 80% population coverage. A Monte Carlo analysis, a
statistical evaluation known in the art, can be employed to assess
the breadth, or redundancy of, population coverage.
[0314] 4.) When selecting epitopes from cancer-related antigens it
is often useful to select analogs because the patient may have
developed tolerance to the native epitope.
[0315] 5.) Of particular relevance are epitopes referred to as
"nested epitopes." Nested epitopes occur where at least two
epitopes overlap in a given peptide sequence. A nested peptide
sequence can comprise B cell, HLA class I and/or HLA class II
epitopes. When providing nested epitopes, a general objective is to
provide the greatest number of epitopes per sequence. Thus, an
aspect is to avoid providing a peptide that is any longer than the
amino terminus of the amino terminal epitope and the carboxyl
terminus of the carboxyl terminal epitope in the peptide. When
providing a multi-epitopic sequence, such as a sequence comprising
nested epitopes, it is generally important to screen the sequence
in order to insure that it does not have pathological or other
deleterious biological properties.
[0316] 6.) If a polyepitopic protein is created, or when creating a
minigene, an objective is to generate the smallest peptide that
encompasses the epitopes of interest. This principle is similar, if
not the same as that employed when selecting a peptide comprising
nested epitopes. However, with an artificial polyepitopic peptide,
the size minimization objective is balanced against the need to
integrate any spacer sequences between epitopes in the polyepitopic
protein. Spacer amino acid residues can, for example, be introduced
to avoid junctional epitopes (an epitope recognized by the immune
system, not present in the target antigen, and only created by the
man-made juxtaposition of epitopes), or to facilitate cleavage
between epitopes and thereby enhance epitope presentation.
Junctional epitopes are generally to be avoided because the
recipient may generate an immune response to that non-native
epitope. Of particular concern is a junctional epitope that is a
"dominant epitope." A dominant epitope may lead to such a zealous
response that immune responses to other epitopes are diminished or
suppressed.
[0317] 7.) Where the sequences of multiple variants of the same
target protein are present, potential peptide epitopes can also be
selected on the basis of their conservancy. For example, a
criterion for conservancy may define that the entire sequence of an
HLA class I binding peptide or the entire 9-mer core of a class II
binding peptide be conserved in a designated percentage of the
sequences evaluated for a specific protein antigen.
[0318] X.C.1. Minigene Vaccines
[0319] A number of different approaches are available which allow
simultaneous delivery of multiple epitopes. Nucleic acids encoding
the peptides of the invention are a particularly useful embodiment
of the invention. Epitopes for inclusion in a minigene are
preferably selected according to the guidelines set forth in the
previous section. A preferred means of administering nucleic acids
encoding the peptides of the invention uses minigene constructs
encoding a peptide comprising one or multiple epitopes of the
invention.
[0320] The use of multi-epitope minigenes is described below and
in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and
Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J.
Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348,
1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a
multi-epitope DNA plasmid encoding supernotif- and/or motif-bearing
epitopes derived 158P1H4, the PADRE.RTM. universal helper T cell
epitope (or multiple HTL epitopes from 158P1H4) and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0321] The immunogenicity of a multi-epitopic minigene can be
confirmed in transgenic mice to evaluate the magnitude of CTL
induction responses against the epitopes tested. Further, the
immunogenicity of DNA-encoded epitopes in vivo can be correlated
with the in vitro responses of specific CTL lines against target
cells transfected with the DNA plasmid. Thus, these experiments can
show that the minigene serves to both: 1.) generate a CTL response
and 2.) that the induced CTLs recognized cells expressing the
encoded epitopes.
[0322] For example, to create a DNA sequence encoding the selected
epitopes (minigene) for expression in human cells, the amino acid
sequences of the epitopes may be reverse translated. A human codon
usage table can be used to guide the codon choice for each amino
acid. These epitope-encoding DNA sequences may be directly
adjoined, so that when translated, a continuous polypeptide
sequence is created. To optimize expression and/or immunogenicity,
additional elements can be incorporated into the minigene design.
Examples of amino acid sequences that can be reverse translated and
included in the minigene sequence include: HLA class I epitopes,
HLA class II epitopes, antibody epitopes, a ubiquitination signal
sequence, and/or an endoplasmic reticulum targeting signal. In
addition, HLA presentation of CTL and HTL epitopes may be improved
by including synthetic (e.g. poly-alanine) or naturally-occurring
flanking sequences adjacent to the CTL or HTL epitopes; these
larger peptides comprising the epitope(s) are within the scope of
the invention.
[0323] The minigene sequence may be converted to DNA by assembling
oligonucleotides that encode the plus and minus strands of the
minigene. Overlapping oligonucleotides (30-100 bases long) may be
synthesized, phosphorylated, purified and annealed under
appropriate conditions using well known techniques. The ends of the
oligonucleotides can be joined, for example, using T4 DNA ligase.
This synthetic minigene, encoding the epitope polypeptide, can then
be cloned into a desired expression vector.
[0324] Standard regulatory sequences well known to those of skill
in the art are preferably included in the vector to ensure
expression in the target cells. Several vector elements are
desirable: a promoter with a down-stream cloning site for minigene
insertion; a polyadenylation signal for efficient transcription
termination; an E. coli origin of replication; and an E. coli
selectable marker (e.g. ampicillin or kanamycin resistance).
Numerous promoters can be used for this purpose, e.g. the human
cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos.
5,580,859 and 5,589,466 for other suitable promoter sequences.
[0325] Additional vector modifications may be desired to optimize
minigene expression and immunogenicity. In some cases, introns are
required for efficient gene expression, and one or more synthetic
or naturally-occurring introns could be incorporated into the
transcribed region of the minigene. The inclusion of mRNA
stabilization sequences and sequences for replication in mammalian
cells may also be considered for increasing minigene
expression.
[0326] Once an expression vector is selected, the minigene is
cloned into the polylinker region downstream of the promoter. This
plasmid is transformed into an appropriate E. coli strain, and DNA
is prepared using standard techniques. The orientation and DNA
sequence of the minigene, as well as all other elements included in
the vector, are confirmed using restriction mapping and DNA
sequence analysis. Bacterial cells harboring the correct plasmid
can be stored as a master cell bank and a working cell bank.
[0327] In addition, immunostimulatory sequences (ISSs or CpGs)
appear to play a role in the immunogenicity of DNA vaccines. These
sequences may be included in the vector, outside the minigene
coding sequence, if desired to enhance immunogenicity.
[0328] In some embodiments, a bi-cistronic expression vector which
allows production of both the minigene-encoded epitopes and a
second protein (included to enhance or decrease immunogenicity) can
be used. Examples of proteins or polypeptides that could
beneficially enhance the immune response if co-expressed include
cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules
(e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR
binding proteins (PADRE.TM., Epimmune, San Diego, Calif.). Helper
(HTL) epitopes can be joined to intracellular targeting signals and
expressed separately from expressed CTL epitopes; this allows
direction of the HTL epitopes to a cell compartment different than
that of the CTL epitopes. If required, this could facilitate more
efficient entry of HTL epitopes into the HLA class II pathway,
thereby improving HTL induction. In contrast to HTL or CTL
induction, specifically decreasing the immune response by
co-expression of immunosuppressive molecules (e.g. TGF-.beta.) may
be beneficial in certain diseases.
[0329] Therapeutic quantities of plasmid DNA can be produced for
example, by fermentation in E. coli, followed by purification.
Aliquots from the working cell bank are used to inoculate growth
medium, and grown to saturation in shaker flasks or a bioreactor
according to well-known techniques. Plasmid DNA can be purified
using standard bioseparation technologies such as solid phase
anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.).
If required, supercoiled DNA can be isolated from the open circular
and linear forms using gel electrophoresis or other methods.
[0330] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite, Bio
Techniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309;
and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In
addition, peptides and compounds referred to collectively as
protective, interactive, non-condensing compounds (PINC) could also
be complexed to purified plasmid DNA to influence variables such as
stability, intramuscular dispersion, or trafficking to specific
organs or cell types.
[0331] Target cell sensitization can be used as a functional assay
for expression and HLA class I presentation of minigene-encoded CTL
epitopes. For example, the plasmid DNA is introduced into a
mammalian cell line that is suitable as a target for standard CTL
chromium release assays. The transfection method used will be
dependent on the final formulation. Electroporation can be used for
"naked" DNA, whereas cationic lipids allow direct in vitro
transfection. A plasmid expressing green fluorescent protein (GFP)
can be co-transfected to allow enrichment of transfected cells
using fluorescence activated cell sorting (FACS). These cells are
then chromium-51 (.sup.51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by .sup.51Cr
release, indicates both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be
evaluated in an analogous manner using assays to assess HTL
activity.
[0332] In vivo immunogenicity is a second approach for functional
testing of minigene DNA formulations. Transgenic mice expressing
appropriate human HLA proteins are immunized with the DNA product.
The dose and route of administration are formulation dependent
(e.g., IM for DNA in PBS, intraperitoneal (i.p.) for
lipid-complexed DNA). Twenty-one days after immunization,
splenocytes are harvested and restimulated for one week in the
presence of peptides encoding each epitope being tested.
Thereafter, for CTL effector cells, assays are conducted for
cytolysis of peptide-loaded, .sup.51Cr-labeled target cells using
standard techniques. Lysis of target cells that were sensitized by
HLA loaded with peptide epitopes, corresponding to minigene-encoded
epitopes, demonstrates DNA vaccine function for in vivo induction
of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic
mice in an analogous manner.
[0333] Alternatively, the nucleic acids can be administered using
ballistic delivery as described, for instance, in U.S. Pat. No.
5,204,253. Using this technique, particles comprised solely of DNA
are administered. In a further alternative embodiment, DNA can be
adhered to particles, such as gold particles.
[0334] Minigenes can also be delivered using other bacterial or
viral delivery systems well known in the art, e.g., an expression
construct encoding epitopes of the invention can be incorporated
into a viral vector such as vaccinia.
[0335] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0336] Vaccine compositions comprising CTL peptides of the
invention can be modified, e.g., analoged, to provide desired
attributes, such as improved serum half life, broadened population
coverage or enhanced immunogenicity.
[0337] For instance, the ability of a peptide to induce CTL
activity can be enhanced by linking the peptide to a sequence which
contains at least one epitope that is capable of inducing a T
helper cell response. Although a CTL peptide can be directly linked
to a T helper peptide, often CTL epitope/HTL epitope conjugates are
linked by a spacer molecule. The spacer is typically comprised of
relatively small, neutral molecules, such as amino acids or amino
acid minetics, which are substantially uncharged under
physiological conditions. The spacers are typically selected from,
e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or
neutral polar amino acids. It will be understood that the
optionally present spacer need not be comprised of the same
residues and thus may be a hetero- or homo-oligomer. When present,
the spacer will usually be at least one or two residues, more
usually three to six residues and sometimes 10 or more residues.
The CTL peptide epitope can be linked to the T helper peptide
epitope either directly or via a spacer either at the amino or
carboxy terminus of the CTL peptide. The amino terminus of either
the immunogenic peptide or the T helper peptide may be
acylated.
[0338] In certain embodiments, the T helper peptide is one that is
recognized by T helper cells present in a majority of a genetically
diverse population. This can be accomplished by selecting peptides
that bind to many, most, or all of the HLA class II molecules.
Examples of such amino acid bind many HLA Class II molecules
include sequences from antigens such as tetanus toxoid at positions
830-843 (QYIKANSKFIGITE; SEQ ID NO: 709), Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 710), and Streptococcus 18kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 711).
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0339] Alternatively, it is possible to prepare synthetic peptides
capable of stimulating T helper lymphocytes, in a loosely
HLA-restricted fashion, using amino acid sequences not found in
nature (see, e.g., PCT publication WO 95/07707). These synthetic
compounds called Pan-DR-binding epitopes (e.g., PADRE.TM.,
Epimmune, Inc., San Diego, Calif.) are designed to most preferably
bind most HLA-DR (human HLA class II) molecules. For instance, a
pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa
(SEQ ID NO: 712), where "X" is either cyclohexylalanine,
phenylalanine, or tyrosine, and a is either D-alanine or L-alanine,
has been found to bind to most HLA-DR alleles, and to stimulate the
response of T helper lymphocytes from most individuals, regardless
of their HLA type. An alternative of a pan-DR binding epitope
comprises all "L" natural amino acids and can be provided in the
form of nucleic acids that encode the epitope.
[0340] HTL peptide epitopes can also be modified to alter their
biological properties. For example, they can be modified to include
D-amino acids to increase their resistance to proteases and thus
extend their serum half life, or they can be conjugated to other
molecules such as lipids, proteins, carbohydrates, and the like to
increase their biological activity. For example, a T helper peptide
can be conjugated to one or more palmitic acid chains at either the
amino or carboxyl termini.
[0341] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0342] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes B lymphocytes or T lymphocytes. Lipids have been
identified as agents capable of priming CTL in vivo. For example,
palmitic acid residues can be attached to the .epsilon.- and
.alpha.-amino groups of a lysine residue and then linked, e.g., via
one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser,
or the like, to an immunogenic peptide. The lipidated peptide can
then be administered either directly in a micelle or particle,
incorporated into a liposome, or emulsified in an adjuvant, e.g.,
incomplete Freund's adjuvant. In a preferred embodiment, a
particularly effective immunogenic composition comprises palmitic
acid attached to .epsilon.- and .alpha.-amino groups of Lys, which
is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the immunogenic peptide.
[0343] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) can be
used to prime virus specific CTL when covalently attached to an
appropriate peptide (see, e.g., Deres, et al., Nature 342:561,
1989). Peptides of the invention can be coupled to P.sub.3CSS, for
example, and the lipopeptide administered to an individual to
specifically prime an immune response to the target antigen.
Moreover, because the induction of neutralizing antibodies can also
be primed with P.sub.3CSS-conjugated epitopes, two such
compositions can be combined to more effectively elicit both
humoral and cell-mediated responses.
[0344] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0345] An embodiment of a vaccine composition in accordance with
the invention comprises ex vivo administration of a cocktail of
epitope-bearing peptides to PBMC, or isolated DC therefrom, from
the patient's blood. A pharmaceutical to facilitate harvesting of
DC can be used, such as Progenipoietin.TM. (Pharmacia-Monsanto, St.
Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and
prior to reinfusion into patients, the DC are washed to remove
unbound peptides. In this embodiment, a vaccine comprises
peptide-pulsed DCs which present the pulsed peptide epitopes
complexed with HLA molecules on their surfaces.
[0346] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 158P1H4. Optionally, a
helper T cell (HTL) peptide, such as a natural or artificial
loosely restricted HLA Class II peptide, can be included to
facilitate the CTL response. Thus, a vaccine in accordance with the
invention is used to treat a cancer which expresses or
overexpresses 158P1H4.
[0347] X.D. Adoptive Immunotherapy
[0348] Antigenic 158P1H4-related peptides are used to elicit a CTL
and/or HTL response ex vivo, as well. The resulting CTL or HTL
cells, can be used to treat tumors in patients that do not respond
to other conventional forms of therapy, or will not respond to a
therapeutic vaccine peptide or nucleic acid in accordance with the
invention. Ex vivo CTL or HTL responses to a particular antigen are
induced by incubating in tissue culture the patient's, or
genetically compatible, CTL or HTL precursor cells together with a
source of antigen-presenting cells (APC), such as dendritic cells,
and the appropriate immmunogenic peptide. After an appropriate
incubation time (typically about 7-28 days), in which the precursor
cells are activated and expanded into effector cells, the cells are
infused back into the patient, where they will destroy (CTL) or
facilitate destruction (HTL) of their specific target cell (e.g., a
tumor cell). Transfected dendritic cells may also be used as
antigen presenting cells.
[0349] X.E. Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0350] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 158P1H4. In therapeutic applications, peptide and/or
nucleic acid compositions are administered to a patient in an
amount sufficient to elicit an effective B cell, CTL and/or HTL
response to the antigen and to cure or at least partially arrest or
slow symptors and/or complications. An amount adequate to
accomplish this is defined as "therapeutically effective dose."
Amounts effective for this use will depend on, e.g., the particular
composition administered, the manner of administration, the stage
and severity of the disease being treated, the weight and general
state of health of the patient, and the judgment of the prescribing
physician.
[0351] For pharmaceutical compositions, the immunogenic peptides of
the invention, or DNA encoding them, are generally administered to
an individual already bearing a tumor that expresses 158P1H4. The
peptides or DNA encoding them can be administered individually or
as fusions of one or more peptide sequences. Patients can be
treated with the immunogenic peptides separately or in conjunction
with other treatments, such as surgery, as appropriate.
[0352] For therapeutic use, administration should generally begin
at the first diagnosis of 158P1H4-associated cancer. This is
followed by boosting doses until at least symptoms are
substantially abated and for a period thereafter. The embodiment of
the vaccine composition (i.e., including, but not limited to
embodiments such as peptide cocktails, polyepitopic polypeptides,
minigenes, or TAA-specific CTLs or pulsed dendritic cells)
delivered to the patient may vary according to the stage of the
disease or the patient's health status. For example, in a patient
with a tumor that expresses 158P1H4, a vaccine comprising
158P1H4-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0353] It is generally important to provide an amount of the
peptide epitope delivered by a mode of administration sufficient to
effectively stimulate a cytotoxic T cell response; compositions
which stimulate helper T cell responses can also be given in
accordance with this embodiment of the invention.
[0354] The dosage for an initial therapeutic immunization generally
occurs in a unit dosage range where the lower value is about 1, 5,
50, 500, or 1,000 .mu.g and the higher value is about 10,000;
20,000; 30,000; or 50,000 .mu.g. Dosage values for a human
typically range from about 500 .mu.g to about 50,000 .mu.g per 70
kilogram patient. Boosting dosages of between about 1.0 .mu.g to
about 50,000 .mu.g of peptide pursuant to a boosting regimen over
weeks to months may be administered depending upon the patients
response and condition as determined by measuring the specific
activity of CTL and HTL obtained from the patient's blood.
Administration should continue until at least clinical symptoms or
laboratory tests indicate that the neoplasia, has been eliminated
or reduced and for a period thereafter. The dosages, routes of
administration, and dose schedules are adjusted in accordance with
methodologies known in the art.
[0355] In certain embodiments, the peptides and compositions of the
present invention are employed in serious disease states, that is,
life-threatening or potentially life threatening situations. In
such cases, as a result of the minimal amounts of extraneous
substances and the relative nontoxic nature of the peptides in
preferred compositions of the invention, it is possible and may be
felt desirable by the treating physician to administer substantial
excesses of these peptide compositions relative to these stated
dosage amounts.
[0356] The vaccine compositions of the invention can also be used
purely as prophylactic agents. Generally the dosage for an initial
prophylactic immunization generally occurs in a unit dosage range
where the lower value is about 1, 5, 50, 500, or 1000 .mu.g and the
higher value is about 10,000; 20,000; 30,000; or 50,000 .mu.g.
Dosage values for a human typically range from about 500 .mu.g to
about 50,000 .mu.g per 70 kilogram patient. This is followed by
boosting dosages of between about 1.0 .mu.g to about 50,000 .mu.g
of peptide administered at defined intervals from about four weeks
to six months after the initial administration of vaccine. The
immunogenicity of the vaccine can be assessed by measuring the
specific activity of CTL and HTL obtained from a sample of the
patient's blood.
[0357] The pharmaceutical compositions for therapeutic treatment
are intended for parenteral, topical, oral, nasal, intrathecal, or
local (e.g. as a cream or topical ointment) administration.
Preferably, the pharmaceutical compositions are administered
parentally, e.g., intravenously, subcutaneously, intradermally, or
intramuscularly. Thus, the invention provides compositions for
parenteral administration which comprise a solution of the
immunogenic peptides dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier.
[0358] A variety of aqueous carriers may be used, e.g., water,
buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the
like. These compositions may be sterilized by conventional,
well-known sterilization techniques, or may be sterile filtered.
The resulting aqueous solutions may be packaged for use as is, or
lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration.
[0359] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservatives, and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
[0360] The concentration of peptides of the invention in the
pharmaceutical formulations can vary widely, i.e., from less than
about 0.1%, usually at or at least about 2% to as much as 20% to
50% or more by weight, and will be selected primarily by fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected.
[0361] A human unit dose form of the peptide composition is
typically included in a pharmaceutical composition that comprises a
human unit dose of an acceptable carrier, preferably an aqueous
carrier, and is administered in a volume of fluid that is known by
those of skill in the art to be used for administration of such
compositions to humans (see, e.g., Remington's Pharmaceutical
Sciences, 17.sup.th Edition, A. Gennaro, Editor, Mack Publishing
Co., Easton, Pa. 1985).
[0362] Proteins(s) of the invention, and/or nucleic acids encoding
the protein(s), can also be administered via liposomes, which may
also serve to: 1) target the proteins(s) to a particular tissue,
such as lymphoid tissue; 2) to target selectively to diseases
cells; or, 3) to increase the half-life of the peptide composition.
Liposomes include emulsions, foams, micelles, insoluble monolayers,
liquid crystals, phospholipid dispersions, lamellar layers and the
like. In these preparations, the peptide to be delivered is
incorporated as part of a liposome, alone or in conjunction with a
molecule which binds to a receptor prevalent among lymphoid cells,
such as monoclonal antibodies which bind to the CD45 antigen, or
with other therapeutic or immunogenic compositions. Thus, liposomes
either filled or decorated with a desired peptide of the invention
can be directed to the site of lymphoid cells, where the liposomes
then deliver the peptide compositions. Liposomes for use in
accordance with the invention are formed from standard
vesicle-forming lipids, which generally include neutral and
negatively charged phospholipids and a sterol, such as cholesterol.
The selection of lipids is generally guided by consideration of,
e.g., liposome size, acid lability and stability of the liposomes
in the blood stream. A variety of methods are available for
preparing liposomes, as described in, e.g., Szoka, et al., Ann.
Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871,
4,501,728, 4,837,028, and 5,019,369.
[0363] For targeting cells of the immune system, a ligand to be
incorporated into the liposome can include, e.g., antibodies or
fragments thereof specific for cell surface determinants of the
desired immune system cells. A liposome suspension containing a
peptide may be administered intravenously, locally, topically, etc.
in a dose which varies according to, inter alia, the manner of
administration, the peptide being delivered, and the stage of the
disease being treated.
[0364] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition is formed by incorporating any of the normally
employed excipients, such as those carriers previously listed, and
generally 10-95% of active ingredient, that is, one or more
peptides of the invention, and more preferably at a concentration
of 25%-75%.
[0365] For aerosol administration, immunogenic peptides are
preferably supplied in finely divided form along with a surfactant
and propellant. Typical percentages of peptides are about 0.01%-20%
by weight, preferably about 1%-10%. The surfactant must, of course,
be nontoxic, and preferably soluble in the propellant.
Representative of such agents are the esters or partial esters of
fatty acids containing from about 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids with an aliphatic polyhydric alcohol or
its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be employed. The surfactant may constitute about
0.1%-20% by weight of the composition, preferably about 0.25-5%.
The balance of the composition is ordinarily propellant. A carrier
can also be included, as desired, as with, e.g., lecithin for
intranasal delivery.
[0366] XI.) Diagnostic and Prognostic Embodiments of 158P1H4
[0367] As disclosed herein, 158P1H4 polynucleotides, polypeptides,
reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and
anti-polypeptide antibodies are used in well known diagnostic,
prognostic and therapeutic assays that examine conditions
associated with dysregulated cell growth such as cancer, in
particular the cancers listed in Table I (see, e.g., both its
specific pattern of tissue expression as well as its overexpression
in certain cancers as described for example in Example 4).
[0368] 158P1H4 can be used in a manner anogous to, or as
complementary to, the bladder associated antigen combination,
mucins and CEA, represented in a diagnostic kit called
InmunoCyt.TM.. ImmunoCyt a is a commercialy available assay to
identify and monitor the presence of bladder cancer (see Fradet et
al., 1997, Can J Urol, 4(3):400-405). A variety of other diagnostic
markers are also used in similar contexts including p53 and H-ras
(see, e.g., Tulchinsky et al., Int J Mol Med July 1999 4(1):99-102
and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12).
Therefore, this disclosure of the 158P1H4 polynucleotides and
polypeptides (as well as the 158P1H4 polynucleotide probes and
anti-158P1H4 antibodies used to identify the presence of these
molecules) and their properties allows skilled artisans to utilize
these molecules in methods that are analogous to those used, for
example, in a variety of diagnostic assays directed to examining
conditions associated with cancer.
[0369] Typical embodiments of diagnostic methods which utilize the
158P1H4 polynucleotides, polypeptides, reactive T cells and
antibodies are analogous to those methods from well-established
diagnostic assays which employ, e.g., PSA polynucleotides,
polypeptides, reactive T cells and antibodies. For example, just as
PSA polynucleotides are used as probes (for example in Northern
analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int.
33(3):567-74(1994)) and primers (for example in PCR analysis, see,
e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe
the presence and/or the level of PSA mRNAs in methods of monitoring
PSA overexpression or the metastasis of prostate cancers, the
158P1H4 polynucleotides described herein can be utilized to detect
158P1H4 overexpression or the metastasis of bladder and other
cancers expressing this gene. Alternatively, just as PSA
polypeptides are used to generate antibodies specific for PSA which
can then be used to observe the presence and/or the level of PSA
proteins in methods to monitor PSA protein overexpression (see,
e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis
of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract.
192(3):233-7 (1996)), the 158P1H4 polypeptides described herein can
be utilized to generate antibodies for use in detecting 158P1H4
overexpression or the metastasis of bladder cells and cells of
other cancers expressing this gene.
[0370] Specifically, because metastases involves the movement of
cancer cells from an organ of origin (such as the lung or bladder
etc.) to a different area of the body (such as a lymph node),
assays which examine a biological sample for the presence of cells
expressing 158P1H4 polynucleotides and/or polypeptides can be used
to provide evidence of metastasis. For example, when a biological
sample from tissue that does not normally contain
158P1H4-expressing cells (lymph node) is found to contain
158P1H4-expressing cells such as the 158P1H4 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0371] Alternatively 158P1H4 polynucleotides and/or polypeptides
can be used to provide evidence of cancer, for example, when cells
in a biological sample that do not normally express 158P1H4 or
express 158P1H4 at a different level are found to express 158P1H4
or have an increased expression of 158P1H4 (see, e.g., the 158P1H4
expression in the cancers listed in Table I and in patient samples
etc. shown in the accompanying Figures). In such assays, artisans
may further wish to generate supplementary evidence of metastasis
by testing the biological sample for the presence of a second
tissue restricted marker (in addition to 158P1H4) such as
ImmunoCyt.TM., PSCA etc. (see, e.g., Fradet et al., 1997, Can J
Urol, 4(3):400-405; Amara et al., 2001, Cancer Res 61:4660-4665 ).
Just as PSA polynucleotide fragments and polynucleotide variants
are employed by skilled artisans for use in methods of monitoring
PSA, 158P1H4 polynucleotide fragments and polynucleotide variants
are used in an analogous manner. In particular, typical PSA
polynucleotides used in methods of monitoring PSA are probes or
primers which consist of fragments of the PSA cDNA sequence.
Illustrating this, primers used to PCR amplify a PSA polynucleotide
must include less than the whole PSA sequence to function in the
polymerase chain reaction. In the context of such PCR reactions,
skilled artisans generally create a variety of different
polynucleotide fragments that can be used as primers in order to
amplify different portions of a polynucleotide of interest or to
optimize amplification reactions (see, e.g., Caetano-Anolles, G.
Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al.,
Methods Mol. Biol. 98:121-154 (1998)). An additional illustration
of the use of such fragments is provided in Example 4, where a
158P1H4 polynucleotide fragment is used as a probe to show the
expression of 158P1H4 RNAs in cancer cells. In addition, variant
polynucleotide sequences are typically used as primers and probes
for the corresponding mRNAs in PCR and Northern analyses (see,
e.g., Sawai et al., Fetal Diagn. Ther. November-December 1996 11
(6):407-13 and Current Protocols In Molecular Biology, Volume 2,
Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide
fragments and variants are useful in this context where they are
capable of binding to a target polynucleotide sequence (e.g. the
158P1H4 polynucleotide shown in SEQ ID NO: 701) under conditions of
high stringency.
[0372] Furthermore, PSA polypeptides which contain an epitope that
can be recognized by an antibody or T cell that specifically binds
to that epitope are used in methods of monitoring PSA. 158P1H4
polypeptide fragments and polypeptide analogs or variants can also
be used in an analogous manner. This practice of using polypeptide
fragments or polypeptide variants to generate antibodies (such as
anti-PSA antibodies or T cells) is typical in the art with a wide
variety of systems such as fusion proteins being used by
practitioners (see, e.g., Current Protocols In Molecular Biology,
Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this
context, each epitope(s) functions to provide the architecture with
which an antibody or T cell is reactive. Typically, skilled
artisans create a variety of different polypeptide fragments that
can be used in order to generate immune responses specific for
different portions of a polypeptide of interest (see, e.g., U.S.
Pat. Nos. 5,840,501 and 5,939,533). For example it may be
preferable to utilize a polypeptide comprising one of the 158P1H4
biological motifs discussed herein or a motif-bearing subsequence
which is readily identified by one of skill in the art based on
motifs available in the art. Polypeptide fragments, variants or
analogs are typically useful in this context as long as they
comprise an epitope capable of generating an antibody or T cell
specific for a target polypeptide sequence (e.g. the 158P1H4
polypeptide shown in SEQ ID NO: 703).
[0373] As shown herein, the 158P1H4 polynucleotides and
polypeptides (as well as the 158P1H4 polynucleotide probes and
anti-158P1H4 antibodies or T cells used to identify the presence of
these molecules) exhibit specific properties that make them useful
in diagnosing cancers such as those listed in Table I. Diagnostic
assays that measure the presence of 158P1H4 gene products, in order
to evaluate the presence or onset of a disease condition described
herein, such as bladder cancer, are used to identify patients for
preventive measures or further monitoring, as has been done so
successfully with PSA for monitoring prostate cancer. Materials
such as 158P1H4 polynucleotides and polypeptides (as well as the
158P1H4 polynucleotide probes and anti-158P1H4 antibodies used to
identify the presence of these molecules) satisfy a need in the art
for molecules having similar or complementary characteristics to
PSA in situations of bladder cancer. Finally, in addition to their
use in diagnostic assays, the 158P1H4 polynucleotides disclosed
herein have a number of other utilities such as their use in the
identification of oncogenetic associated chromosomal abnormalities
in the chromosomal region to which the 158P1H4 gene maps (see
Example 3 below). Moreover, in addition to their use in diagnostic
assays, the 158P1H4-related proteins and polynucleotides disclosed
herein have other utilities such as their use in the forensic
analysis of tissues of unknown origin (see, e.g., Takahama K
Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).
[0374] Additionally, 158P1H4-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 158P1H4. For example, the
amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or
fragments of either, can be used to generate an immune response to
the 158P1H4 antigen. Antibodies or other molecules that react with
158P1H4 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0375] XII.) Inhibition of 158P1H4 Protein Function
[0376] The invention includes various methods and compositions for
inhibiting the binding of 158P1H4 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
158P1H4 function.
[0377] XII.A.) Inhibition of 158P1H4 with Intracellular
Antibodies
[0378] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 158P1H4 are introduced
into 158P1H4 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-158P1H4 antibody is
expressed intracellularly, binds to 158P1H4 protein, and thereby
inhibits its function. Methods for engineering such intracellular
single chain antibodies are well known. Such intracellular
antibodies, also known as "intrabodies", are specifically targeted
to a particular compartment within the cell, providing control over
where the inhibitory activity of the treatment is focused. This
technology has been successfully applied in the art (for review,
see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies
have been shown to virtually eliminate the expression of otherwise
abundant cell surface receptors (see, e.g., Richardson et al.,
1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,
1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene
Ther. 1: 332-337).
[0379] Single chain antibodies comprise the variable domains of the
heavy and light chain joined by a flexible linker polypeptide, and
are expressed as a single polypeptide. Optionally, single chain
antibodies are expressed as a single chain variable region fragment
joined to the light chain constant region. Well-known intracellular
trafficking signals are engineered into recombinant polynucleotide
vectors encoding such single chain antibodies in order to precisely
target the intrabody to the desired intracellular compartment. For
example, intrabodies targeted to the endoplasmic reticulum (ER) are
engineered to incorporate a leader peptide and, optionally, a
C-terminal ER retention signal, such as the KDEL amino acid motif.
Intrabodies intended to exert activity in the nucleus are
engineered to include a nuclear localization signal. Lipid moieties
are joined to intrabodies in order to tether the intrabody to the
cytosolic side of the plasma membrane. Intrabodies can also be
targeted to exert function in the cytosol. For example, cytosolic
intrabodies are used to sequester factors within the cytosol,
thereby preventing them from being transported to their natural
cellular destination.
[0380] In one embodiment, intrabodies are used to capture 158P1H4
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals are engineered into such 158P1H4
intrabodies in order to achieve the desired targeting. Such 158P1H4
intrabodies are designed to bind specifically to a particular
158P1H4 domain. In another embodiment, cytosolic intrabodies that
specifically bind to the 158P1H4 protein are used to prevent
158P1H4 from gaining access to the nucleus, thereby preventing it
from exerting any biological activity within the nucleus (e.g.,
preventing 158P1H4 from forming transcription complexes with other
factors).
[0381] In order to specifically direct the expression of such
intrabodies to particular cells, the transcription of the intrabody
is placed under the regulatory control of an appropriate
tumor-specific promoter and/or enhancer. In order to target
intrabody expression specifically to bladder, for example, the PSCA
promoter and/or promoter/enhancer can be utilized (See, for
example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999 and Lin et al.
PNAS, USA 92(3):679-683 (1995)).
[0382] XII.B.) Inhibition of 158P1H4 with Recombinant Proteins
[0383] In another approach, recombinant molecules bind to 158P1H4
and thereby inhibit 158P1H4 function. For example, these
recombinant molecules prevent or inhibit 158P1H4 from
accessing/binding to its binding partner(s) or associating with
other protein(s). Such recombinant molecules can, for example,
contain the reactive part(s) of a 158P1H4 specific antibody
molecule. In a particular embodiment, the 158P1H4 binding domain of
a 158P1H4 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 158P1H4 ligand
binding domains linked to the Fc portion of a human IgG, such as
human IgG1. Such IgG portion can contain, for example, the C.sub.H2
and C.sub.H3 domains and the hinge region, but not the C.sub.H1
domain. Such dimeric fusion proteins are administered in soluble
form to patients suffering from a cancer associated with the
expression of 158P1H4, whereby the dimeric fusion protein
specifically binds to 158P1H4 and blocks 158P1H4 interaction with a
binding partner. Such dimeric fusion proteins are further combined
into multimeric proteins using known antibody linking
technologies.
[0384] XII.C.) Inhibition of 158P1H4 Transcription or
Translation
[0385] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 158P1H4 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 158P1H4 mRNA into protein.
[0386] In one approach, a method of inhibiting the transcription of
the 158P1H4 gene comprises contacting the 158P1H4 gene with a
158P1H4 antisense polynucleotide. In another approach, a method of
inhibiting 158P1H4 mRNA translation comprises contacting the
158P1H4 mRNA with an antisense polynucleotide. In another approach,
a 158P1H4 specific ribozyme is used to cleave the 158P1H4 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
158P1H4 gene, such as the 158P1H4 promoter and/or enhancer
elements. Similarly, proteins capable of inhibiting a 158P1H4 gene
transcription factor are used to inhibit 158P1H4 mRNA
transcription. The various polynucleotides and compositions useful
in the aforementioned methods have been described above. The use of
antisense and ribozyme molecules to inhibit transcription and
translation is well known in the art.
[0387] Other factors that inhibit the transcription of 158P1H4 by
interfering with 158P1H4 transcriptional activation are also useful
to treat cancers expressing 158P1H4. Similarly, factors that
interfere with 158P1H4 processing are useful to treat cancers that
express 158P1H4. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0388] XII.D.) General Considerations for Therapeutic
Strategies
[0389] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 158P1H4 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 158P1H4 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 158P1H4 antisense polynucleotides, ribozymes,
factors capable of interfering with 158P1H4 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0390] The above therapeutic approaches can be combined with any
one of a wide variety of surgical, chemotherapy or radiation
therapy regimens. The therapeutic approaches of the invention can
enable the use of reduced dosages of chemotherapy (or other
therapies) and/or less frequent administration, an advantage for
all patients and particularly for those that do not tolerate the
toxicity of the chemotherapeutic agent well.
[0391] The anti-tumor activity of a particular composition (e.g.,
antisense, ribozyme, intrabody), or a combination of such
compositions, can be evaluated using various in vitro and in vivo
assay systems. In vitro assays that evaluate therapeutic activity
include cell growth assays, soft agar assays and other assays
indicative of tumor promoting activity, binding assays capable of
determining the extent to which a therapeutic composition will
inhibit the binding of 158P1H4 to a binding partner, etc.
[0392] In vivo, the effect of a 158P1H4 therapeutic composition can
be evaluated in a suitable animal model. For example, xenogenic
bladder cancer models can be used, wherein human bladder cancer
explants or passaged xenograft tissues are introduced into immune
compromised animals, such as nude or SCID mice (Shibayama et al.,
1991, J Urol., 146(4):1136-7; Beecken et al., 2000, Urology,
56(3):521-526). Efficacy can be predicted using assays that measure
inhibition of tumor formation, tumor regression or metastasis, and
the like.
[0393] In vivo assays that evaluate the promotion of apoptosis are
useful in evaluating therapeutic compositions. In one embodiment,
xenografts from tumor bearing mice treated with the therapeutic
composition can be examined for the presence of apoptotic foci and
compared to untreated control xenograft-bearing mice. The extent to
which apoptotic foci are found in the tumors of the treated mice
provides an indication of the therapeutic efficacy of the
composition.
[0394] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th
Edition, A. Osal., Ed., 1980).
[0395] Therapeutic formulations can be solubilized and administered
via any route capable of delivering the therapeutic composition to
the tumor site. Potentially effective routes of administration
include, but are not limited to, intravenous, parenteral,
intraperitoneal, intramuscular, intratumor, intradermal,
intraorgan, orthotopic, and the like. A preferred formulation for
intravenous injection comprises the therapeutic composition in a
solution of preserved bacteriostatic water, sterile unpreserved
water, and/or diluted in polyvinylchloride or polyethylene bags
containing 0.9% sterile Sodium Chloride for Injection, USP.
Therapeutic protein preparations can be lyophilized and stored as
sterile powders, preferably under vacuum, and then reconstituted in
bacteriostatic water (containing for example, benzyl alcohol
preservative) or in sterile water prior to injection.
[0396] Dosages and administration protocols for the treatment of
cancers using the foregoing methods will vary with the method and
the target cancer, and will generally depend on a number of other
factors appreciated in the art.
[0397] XIII.) Kits
[0398] For use in the diagnostic and therapeutic applications
described herein, kits are also within the scope of the invention.
Such kits can comprise a carrier, package or container that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) comprising one of the
separate elements to be used in the method. For example, the
container(s) can comprise a probe that is or can be detectably
labeled. Such probe can be an antibody or polynucleotide specific
for a 158P1H4-related protein or a 158P1H4 gene or message,
respectively. Where the method utilizes nucleic acid hybridization
to detect the target nucleic acid, the kit can also have containers
containing nucleotide(s) for amplification of the target nucleic
acid sequence and/or a container comprising a reporter-means, such
as a biotin-binding protein, such as avidin or streptavidin, bound
to a reporter molecule, such as an enzymatic, florescent, or
radioisotope label. The kit can include all or part of the amino
acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic
acid molecules that encodes such amino acid sequences.
[0399] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0400] A label can be present on the container to indicate that the
composition is used for a specific therapy or non-therapeutic
application, and can also indicate directions for either in vivo or
in vitro use, such as those described above. Directions and or
other information can also be included on a label or on an insert
which is included with the kit.
EXAMPLES
[0401] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which are intended to limit the scope of the invention.
Example 1
[0402] SSH-Generated Isolation of a cDNA Fragment of the 158P1H4
Gene
[0403] To isolate genes that are over-expressed in bladder cancer
we used the Suppression Subtractive Hybridization (SSH) procedure
using cDNA derived from bladder cancer tissues, including invasive
transitional cell carcinoma. The 158P1H4 SSH cDNA sequence was
derived from a bladder cancer pool minus normal bladder cDNA
subtraction. Included in the driver were also cDNAs derived from
nine other normal tissues. The 158P1H4 cDNA was identified as
highly expressed in the bladder cancer tissue pool, with lower
expression seen in a restricted set of normal tissues.
[0404] The SSH DNA sequence of 90 bp (FIG. 1) has homology to a
chromosome 8q23 Bacterial Artificial Chromosome (BAC) clone
(GenBank accession AP000424) and to an EST (GenBank accession
N95691) derived from a multiple sclerosis library. A 158P1H4 cDNA
clone (clone A) of 2,638 bp was isolated from a human normal
prostate cDNA library, revealing an ORF of 440 amino acids (FIG. 2
and FIG. 3). The nucleotide sequence of 158P1H4 shows homology to
the 2,353 bp sequence (GenBank accession AK014536) encoding the
mouse orthologue of 158P1H4 isolated from mouse day 0 neonatal skin
(FIG. 4).
[0405] The 158P1H4 protein is predicted to be cytoplasmic using the
the PSORT-I program (URL psort.nibb.ac.jp:8800/form.html). Amino
acid sequence analysis of 158P1H4 reveals 75% identity over 438
amino acid region to mouse putative protein (GenBank Accession
BAB29419, FIG. 5A). The highest homology to a known human gene is
seen to sorting nexin 17with 43% identity over 387 amino acids
(GenBank Accession XP.sub.--033201, FIG. 5B).
[0406] Materials and Methods
[0407] Human Tissues
[0408] The bladder cancer patient tissues were purchased from
several sources such as from the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues were purchased from Clontech, Palo Alto,
Calif.
[0409] RNA Isolation
[0410] Tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA.
Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA
Mini and Midi kits. Total and mRNA were quantified by
spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel
electrophoresis.
[0411] Oligonucleotides
[0412] The following HPLC purified oligonucleotides were used.
1 DPNCDN (cDNA synthesis primer): 5'TTTTGATCAAGCTT.sub.303' (SEQ ID
NO:717) Adaptor 1: 5'CTAATACGACTCACTATAGGG- CTCGAGCG (SEQ ID
NO:718) GCCGCCCGGGCAG3' 3'GGCCCGTCCTAG5' (SEQ ID NO:719) Adaptor 2:
5'GTAATACGACTCACTATAGGG- CAGCGTGG (SEQ ID NO:720) TCGCGGCCGAG3'
3'CGGCTCCTAG5' (SEQ ID NO:721) PCR primer 1:
5'CTAATACGACTCACTATAGGGC3- ' (SEQ ID NO:722) Nested primer (NP)1:
5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO:723) Nested primer (NP)2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO:724)
[0413] Suppression Subtractive Hybridization
[0414] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in bladder cancer. The SSH reaction utilized cDNA from
bladder cancer and normal tissues.
[0415] The gene 158P1H4 sequence was derived from a bladder cancer
pool minus normal bladder cDNA subtraction. The SSH DNA sequence
(FIG. 1) was identified.
[0416] The cDNA derived from of pool of normal bladder tissues was
used as the source of the "driver" cDNA, while the cDNA from a pool
of bladder cancer tissues was used as the source of the "tester"
cDNA. Double stranded cDNAs corresponding to tester and driver
cDNAs were synthesized from 2 .mu.g of poly(A).sup.+ RNA isolated
from the relevant xenograft tissue, as described above, using
CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of
oligonucleotide DPNCDN as primer. First- and second-strand
synthesis were carried out as described in the Kit's user manual
protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The
resulting cDNA was digested with Dpn II for 3 hrs at 37.degree. C.
Digested cDNA was extracted with phenol/chloroform (1:1) and
ethanol precipitated.
[0417] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from the relevant tissue source (see above) with a
mix of digested cDNAs derived from the nine normal tissues:
stomach, skeletal muscle, lung, brain, liver, kidney, pancreas,
small intestine, and heart.
[0418] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l, 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH).
Ligation was terminated with 1 .mu.l of 0.2 M EDTA and heating at
72.degree. C. for 5 min.
[0419] The first hybridization was performed by adding 1.5 .mu.l
(600 ng) of driver cDNA to each of two tubes containing 1.5 .mu.l
(20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final
volume of 4 .mu.l, the samples were overlaid with mineral oil,
denatured in an MJ Research thermal cycler at 98.degree. C. for 1.5
minutes, and then were allowed to hybridize for 8 hrs at 68.degree.
C. The two hybridizations were then mixed together with an
additional 1 .mu.l of fresh denatured driver cDNA and were allowed
to hybridize overnight at 68.degree. C. The second hybridization
was then diluted in 200 .mu.l of 20 mM Hepes, pH 8.3, 50 mM NaCl,
0.2 mM EDTA, heated at 70.degree. C. for 7 min. and stored at
-20.degree. C.
[0420] PCR Amplification Cloning and Sequencing of Gene Fragments
Generated from SSH
[0421] To amplify gene fragments resulting from SSH reactions, two
PCR amplifications were performed. In the primary PCR reaction 1
.mu.l of the diluted final hybridization mix was added to 1 .mu.l
of PCR primer 1 (10 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5
.mu.l 10.times.reaction buffer (CLONTECH) and 0.5 .mu.l
50.times.Advantage cDNA polymerase Mix (CLONTECH) in a final volume
of 25 .mu.l. PCR 1 was conducted using the following conditions:
75.degree. C. for 5 min., 94.degree. C. for 25 sec., then 27 cycles
of 94.degree. C. for 10 sec, 66.degree. C. for 30 sec, 72.degree.
C. for 1.5 min. Five separate primary PCR reactions were performed
for each experiment. The products were pooled and diluted 1:10 with
water. For the secondary PCR reaction, 1 .mu.l from the pooled and
diluted primary PCR reaction was added to the same reaction mix as
used for PCR 1, except that primers NP1 and NP2 (10 .mu.M) were
used instead of PCR primer 1. PCR 2 was performed using 10-12
cycles of 94.degree. C. for 10 sec, 68.degree. C. for 30 sec, and
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0422] The PCR products were inserted into pCR2.1 using the TIA
vector cloning kit (Invitrogen). Transformed E. coli were subjected
to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in liquid culture
overnight. To identify inserts, PCR amplification was performed on
1 ml of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0423] Bacterial clones were stored in 20% glycerol in a 96 well
format. Plasmid DNA was prepared, sequenced, and subjected to
nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP
databases.
[0424] RT-PCR Expression Analysis
[0425] First strand cDNAs can be generated from 1 .mu.g of mRNA
with oligo (dT) 12-18 priming using the Gibco-BRL Superscript
Preamplification system. The manufacturer's protocol was used which
included an incubation for 50 min at 42.degree. C. with reverse
transcriptase followed by RNAse H treatment at 37.degree. C. for 20
min. After completing the reaction, the volume can be increased to
200 .mu.l with water prior to normalization. First strand cDNAs
from 16 different normal human tissues can be obtained from
Clontech.
[0426] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 725) and
5'agccacacgcagctcattgtagaagg 3'(SEQ ID NO: 726) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1.times.PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM
MgCl.sub.2, 50 mM KCl, pH8.3) and 1.times.Klentaq DNA polymerase
(Clontech). Five .mu.l of the PCR reaction can be removed at 18,
20, and 22 cycles and used for agarose gel electrophoresis. PCR was
performed using an MJ Research thermal cycler under the following
conditions: Initial denaturation can be at 94.degree. C. for 15
sec, followed by a 18, 20, and 22 cycles of 94.degree. C. for 15,
65.degree. C for 2 min, 72.degree. C. for 5 sec. A final extension
at 72.degree. C. was carried out for 2 min. After agarose gel
electrophoresis, the band intensities of the 283 b.p. .beta.-actin
bands from multiple tissues were compared by visual inspection.
Dilution factors for the first strand cDNAs were calculated to
result in equal .beta.-actin band intensities in all tissues after
22 cycles of PCR. Three rounds of normalization can be required to
achieve equal band intensities in all tissues after 22 cycles of
PCR.
[0427] To determine expression levels of the 158P1H4 gene, 5 .mu.l
of normalized first strand cDNA were analyzed by PCR using 26, and
30 cycles of amplification. Semi-quantitative expression analysis
can be achieved by comparing the PCR products at cycle numbers that
give light band intensities. The primers used for RT-PCR were
designed using the 158P1H4-homologous EST (GenBank accession
N95691) and are listed below:
2 158P1H4.1 5'GTCTCATAAATGACAAGTCGGCAAA3' (SEQ ID NO:727) 158P1H4.2
5'TTTCTCCAGAGCCCTATTCTCCTC3' (SEQ ID NO:728)
[0428] A typical RT-PCR expression analysis is shown in FIG. 6.
RT-PCR expression analysis was performed on first strand cDNAs
generated using pools of tissues from multiple samples. The cDNAs
were shown to be normalized using beta-actin and GADPH PCR.
Expression of 158P1H4 was observed in bladder cancer pool.
Example 2
[0429] Full Length Cloning of 158P1H4
[0430] The 158P1H4 SSH cDNA sequence was derived from a bladder
cancer pool minus normal bladder cDNA subtraction. The SSH cDNA
sequence (FIG. 1) was designated 158P1H4. The full-length cDNA
clone 158P1H4-clone A (FIG. 2) was cloned from a prostate cDNA
library (pEAK8 vector, Pangene) using the 158P1H4 SSH-derived
sequence.
[0431] 158P1H4 clone cDNA was deposited under the terms of the
Budapest Treaty on Mar. 1, 2001, with the American Type Culture
Collection (A.T.C.C.; 10801 University Blvd., Manassas, Va.
20110-2209 USA) as plasmid 158P1H4 A, and has been assigned
Designation No. PTA-3136.
Example 3
[0432] Chromosomal Mapping of 158P1H4
[0433] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are available
including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics
7:22; Research Genetics, Huntsville Ala.), human-rodent somatic
cell hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI Bethesda, Md.).
[0434] 158P1H4 maps to chromosme 8q22-q23, using 158P1H4 sequence
and the NCBI BLAST tool:
(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBla-
st.html&&ORG=Hs). This is a region of frequent
amplification in bladder cancer (Prat et al., Urology May
2001;57(5):986-92; Muscheck et al., Carcinogenesis September
2000;21(9):1721-26) and is associated with rapid tumor cell
proliferation in advanced bladder cancer (Tomovska et al., Int J
Oncol June 2001;18(6):1239-44).
Example 4
[0435] Expression Analysis of 158P1H4 in Normal Tissues and Patient
Specimens
[0436] Analysis by RT-PCR demonstrates that 158P1H4 expression is
restricted to bladder cancer samples (FIG. 6). First strand cDNA
was prepared from VP1 pool (liver, kidney, and lung), VP2 pool
(stomach, pancreas, colon), LAPC xenograft pool (LAPC-4AD,
LAPC-4AI, LAPC-9AD and LAPC-9AI), bladder cancer pool, and cancer
metastasis pool (breast, prostate, bladder, ovarian, pancreatic,
and colon cancer metastasis). Normalization was performed by PCR,
using primers to actin. Semi-quantitative PCR, using primers to
158P1H4 was performed at 26 and 30 cycles of amplification.
Expression of 158P1H4 is observed in bladder cancer pool and in the
metastasis pool but not in normal tissues indicating that 158P1H4
serves as a bladder tumor marker.
[0437] Extensive Northern blot analysis of 158P1H4 in 16 human
normal tissues confirms the expression observed by RT-PCR (FIG. 7).
Even at high exposure, no expression is detected in the 16 normal
tissues tested.
[0438] Northern blot analysis shows that 158P1H4 is expressed in
bladder tumor tissues derived from bladder cancer patients and in 1
out of 3 normal adjacent tissue (FIG. 8). This indicates that
158P1H4 represents a suitable cancer target for cancer diagnosis
and therapy.
Example
[0439] Production of Recombinant 158P1H4 in Prokarvotic Systems
[0440] A. In vitro Transcription and Translation Constructs
[0441] pCRII: To generate 158P1H4 sense and anti-sense RNA probes
for RNA in situ investigations, pCRII constructs (Invitrogen,
Carlsbad Calif.) are generated encoding either all or fragments of
the 158P1H4 cDNA. The pCRII vector has Sp6 and T7 promoters
flanking the insert to drive the transcription of 158P1H4 RNA for
use as probes in RNA in situ hybridization experiments. These
probes are used to analyze the cell and tissue expression of
158P1H4 at the RNA level. Transcribed 158P1H4 RNA representing the
cDNA amino acid coding region of the 158P1H4 gene is used in in
vitro translation systems such as the TnT.TM. Coupled
Reticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize
158P1H4 protein.
[0442] B. Bacterial Constructs
[0443] pGEX Constructs: To generate recombinant 158P1H4 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the 158P1H4 cDNA protein coding sequence
are fused to the GST gene by cloning into pGEX-6P-1 or any other
GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech,
Piscataway, N.J.). These constructs allow controlled expression of
recombinant 158P1H4 protein sequences with GST fused at the
amino-terminus and a six histidine epitope (6.times.His) at the
carboxyl-terminus. The GST and 6.times.His tags permit purification
of the recombinant fusion protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-GST and anti-His antibodies. The 6.times.His tag
is generated by adding 6 histidine codons to the cloning primer at
the 3' end of the open reading frame (ORF). A proteolytic cleavage
site, such as the PreScission.TM. recognition site in pGEX-6P1, may
be employed such that it permits cleavage of the GST tag from
158P1H4-related protein. The ampicillin resistance gene and pBR322
origin permits selection and maintenance of the pGEX plasmids in E.
coli. For example, constructs are made utilizing pGEX-6P-1 such
that the following regions of 158P1H4 are expressed as
amino-terminal fusions to GST: amino acids 1 to 440; or any 8, 9,
10, 11, 12, 13, 14, 15, or more contiguous amino acids from 158P1H4
or analogs thereof.
[0444] pMAL Constructs: To generate recombinant 158P1H4 proteins in
bacterial that are fused to maltose-binding protein (MBP), all or
parts of the 158P1H4 cDNA protein coding sequence are fused to the
MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New
England Biolabs, Beverly, Mass.). These constructs allow controlled
expression of recombinant 158P1H4 protein sequences with MBP fused
at the amino-terminus and a 6.times.His epitope tag at the
carboxyl-terminus. The MBP and 6.times.His tags permit purification
of the recombinant protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-MBP and anti-His antibodies. The 6.times.His
epitope tag is generated by adding 6 histidine codons to the 3'
cloning primer. A Factor Xa recognition site permits cleavage of
the pMAL tag from 158P1H4. The pMAL-c2X and pMAL-p2X vectors are
optimized to express the recombinant protein in the cytoplasm or
periplasm respectively. Periplasm expression enhances folding of
proteins with disulfide bonds. For example, constructs are made
utilizing pMAL-c2X and pMAL-p2X such that the following regions of
the 158P1H4 protein are expressed as amino-terminal fusions to MBP:
amino acids 1 to 440; or any 8, 9, 10, 11, 12,13, 14, 15, or more
contiguous amino acids from 158P1H4 or analogs thereof.
[0445] pET Constructs: To express 158P1H4 in bacterial cells, all
or parts of the 158P1H4 cDNA protein coding sequence are cloned
into the pET family of vectors (Novagen, Madison, Wis.). These
vectors allow tightly controlled expression of recombinant 158P1H4
protein in bacteria with and without fusion to proteins that
enhance solubility, such as NusA and thioredoxin (Trx), and epitope
tags, such as 6.times.His and S-Tag.TM. that aid purification and
detection of the recombinant protein. For example, constructs are
made utilizing pET NusA fusion system 43.1 such that the following
regions of the 158P1H4 protein are expressed as amino-terminal
fusions to NusA: amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13,
14, 15, or more contiguous amino acids from 158P1H4 or analogs
thereof.
[0446] C. Yeast Constructs
[0447] PESC Constructs: To express 158P1H4 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 158P1H4 cDNA protein coding
sequence are cloned into the pESC family of vectors each of which
contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3
(Stratagene, La Jolla, Calif.). These vectors allow controlled
expression from the same plasmid of up to 2 different genes or
cloned sequences containing either Flag.TM. or Myc epitope tags in
the same yeast cell. This system is useful to confirm
protein-protein interactions of 158P1H4. In addition, expression in
yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations, that are found when expressed
in eukaryotic cells. For example, constructs are made utilizing
pESC-HIS such that the following regions of the 158P1H4 protein are
expressed: amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13, 14,
15, or more contiguous amino acids from 158P1H4 or analogs
thereof.
[0448] pESP Constructs: To express 158P1H4 in the yeast species
Saccharomyces pombe, all or parts of the 158P1H4 cDNA protein
coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of expression of a 158P1H4
protein sequence that is fused at either the amino terminus or at
the carboxyl terminus to GST which aids purification of the
recombinant protein. A Flag.TM. epitope tag allows detection of the
recombinant protein with anti-Flag.TM. antibody. For example,
constructs are made utilizing pESP-1 vector such that the following
regions of the 158P1H4 protein are expressed as amino-terminal
fusions to GST: amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13,
14, 15, or more contiguous amino acids from 158P1H4 or analogs
thereof.
Example 6
[0449] Production of Recombinant 158P1H4 in Eukaryotic Systems
[0450] A. Mammalian Constructs
[0451] To express recombinant 158P1H4 in eukaryotic cells, the full
or partial length 158P1H4 cDNA sequences can be cloned into any one
of a variety of expression vectors known in the art. The constructs
can be transfected into any one of a wide variety of mammalian
cells such as 293T cells. Transfected 293T cell lysates can be
probed with the anti-158P1H4 polyclonal serum, described above.
[0452] pcDNA4/HisMax Constructs: To express 158P1H4 in mammalian
cells, the 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 are cloned into pcDNA4/HisMax
Version A (Invitrogen, Carlsbad, Calif.). Protein expression is
driven from the cytomegalovirus (CMV) promoter and the SP163
translational enhancer. The recombinant protein has Xpress.TM. and
six histidine (6.times.His) epitopes fused to the amino-terminus.
The pcDNA4/HisMax vector also contains the bovine growth hormone
(BGH) polyadenylation signal and transcription termination sequence
to enhance mRNA stability along with the SV40 origin for episomal
replication and simple vector rescue in cell lines expressing the
large T antigen. The Zeocin resistance gene allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli. The following regions of 158P1H4 are
expressed in this construct, amino acids 1 to 440; or any 8, 9, 10,
11, 12, 13, 14,15, or more contiguous amino acids from 158P1H4,
variants, or analogs thereof.
[0453] pcDNA3.1/MycHis Constructs: To express 158P1H4 in mammalian
cells, the 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 with a consensus Kozak
translation initiation site are cloned into pcDNA3.1/MycHis Version
A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from
the cytomegalovirus (CMV) promoter. The recombinant proteins have
the myc epitope and 6.times.His epitope fused to the
carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the
bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and Coe1E1 origin permits selection and maintenance
of the plasmid in E. coli. The following regions of 158P1H4 are
expressed in this construct, amino acids 1 to 440; or any 8, 9, 10,
11, 12, 13, 14,15, or more contiguous amino acids from 158P1H4,
variants, or analogs thereof.
[0454] pcDNA3.1/CT-GFP-TOPO Construct: To express 158P1H4 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, the 158P1H4 ORF or sequences coding for a
stretch of contiguous amino acids of 158P1H4 with a consensus Kozak
translation initiation site are cloned into pcDNA3./CT-GFP-TOPO
(Invitrogen, Calif.). Protein expression is driven from the
cytomegalovirus (CMV) promoter. The recombinant proteins have the
Green Fluorescent Protein (GFP) fused to the carboxyl-terminus
facilitating non-invasive, in vivo detection and cell biology
studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine
growth hormone (BGH) polyadenylation signal and transcription
termination sequence to enhance mRNA stability along with the SV40
origin for episomal replication and simple vector rescue in cell
lines expressing the large T antigen. The Neomycin resistance gene
allows for selection of mammalian cells that express the protein,
and the ampicillin resistance gene and ColE1 origin permits
selection and maintenance of the plasmid in E. coli. Additional
constructs with an amino-terminal GFP fusion are made in
pcDNA3.1/NT-GFP-TOPO spanning the entire length of the 158P1H4
proteins. The following regions of 158P1H4 are expressed in these
contructs, amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13,
14,15, or more contiguous amino acids from 158P1H4, variants, or
analogs thereof.
[0455] PAPtag: The 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 are cloned into pAPtag-5
(GenHunter Corp. Nashville, Tenn.). This construct generates an
alkaline phosphatase fusion at the carboxyl-terminus of the 158P1H4
proteins while fusing the IgG.kappa. signal sequence to the
amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgGK signal sequence is fused to
the amino-terminus of 158P2H4 proteins. The resulting recombinant
158P1H4 proteins are optimized for secretion into the media of
transfected mammalian cells and can be used to identify proteins
such as ligands or receptors that interact with the 158P1H4
proteins. Protein expression is driven from the CMV promoter and
the recombinant proteins also contain myc and 6.times.His epitopes
fused at the carboxyl-terminus that facilitates detection and
purification. The Zeocin resistance gene present in the vector
allows for selection of mammalian cells expressing the recombinant
protein and the ampicillin resistance gene permits selection of the
plasmid in E. coli. The following regions of 158P1H4 are expressed
in these constructs, amino acids 1 to 440; or any 8, 9, 10, 11, 12,
13, 14, 15, or more contiguous amino acids from 158P1H4, variants,
or analogs thereof.
[0456] ptag5: The 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 are also cloned into pTag-5. This
vector is similar to pAPtag but without the alkaline phosphatase
fusion. This construct generates 158P1H4 proteins with an
amino-terminal IgG.kappa. signal sequence and myc and 6.times.His
epitope tags at the carboxyl-terminus that facilitate detection and
affinity purification. The resulting recombinant 158P1H4 proteins
are optimized for secretion into the media of transfected mammalian
cells, and can be used as immunogens or ligands to identify
proteins such as ligands or receptors that interact with the
158P1H4 proteins. Protein expression is driven from the CMV
promoter. The Zeocin resistance gene present in the vector allows
for selection of mammalian cells expressing the protein, and the
ampicillin resistance gene permits selection of the plasmid in E.
coli. The following regions of 158P1H4 are expressed in these
constructs, amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13, 14,
15, or more contiguous amino acids from 158P1H4, variants, or
analogs thereof.
[0457] PsecFc: The 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 are also cloned into psecFc. The
psecFc vector was assembled by cloning the human immunoglobulin G1
(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,
California). This construct generates an IgG1 Fc fusion at the
carboxyl-terminus of the 158P1 H4 proteins, while fusing the IgGK
signal sequence to N-terminus. 158P1H4 fusions utilizing the murine
IgG1 Fc region are also used. The resulting recombinant 158P1H4
proteins are optimized for secretion into the media of transfected
mammalian cells, and can be used as immunogens or to identify
proteins such as ligands or receptors that interact with the
158P1H4 protein. Protein expression is driven from the CMV
promoter. The hygromycin resistance gene present in the vector
allows for selection of mammalian cells that express the
recombinant protein, and the ampicillin resistance gene permits
selection of the plasmid in E. coli. The following regions of
158P1H4 are expressed in these constructs, amino acids 1 to 440; or
any 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids
from 158P1H4, variants, or analogs thereof.
[0458] pSR.alpha. Constructs: To generate mammalian cell lines that
express 158P1H4 constitutively, the ORF or sequences coding for a
stretch of contiguous amino acids of 158P1H4 are cloned into
pSR.alpha. constructs. Amphotropic and ecotropic retroviruses are
generated by transfection of pSR.alpha. constructs into the
293T-10A1 packaging line or co-transfection of pSR.alpha. and a
helper plasmid (containing deleted packaging sequences) into the
293 cells, respectively. The retrovirus can be used to infect a
variety of mammalian cell lines, resulting in the integration of
the cloned gene, 158P1H4, into the host cell-lines. Protein
expression is driven from a long terminal repeat (LTR). The
Neomycin resistance gene present in the vector allows for selection
of mammalian cells that express the protein, and the ampicillin
resistance gene and ColE1 origin permit selection and maintenance
of the plasmid in E. coli. The retroviral vectors can thereafter be
used for infection and generation of various cell lines using, for
example, SCaBER, NIH 3T3, TsuPr1, 293 or rat-1 cells.
[0459] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
158P1H4 sequences to allow detection using anti-Flag antibodies.
For example, the FLAG.TM. sequence 5' gat tac aag gat gac gac gat
aag 3' is added to cloning primer at the 3' end of the ORF.
Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length 158P1 H4 proteins. The following
regions of 158P1 H4 expressed in such constructs, amino acids 1 to
440; or any 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino
acids from 158P1H4, variants, or analogs thereof.
[0460] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 158P1H4. High virus titer
leading to high level expression of 158P1H4 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. The 158P1H4 coding sequences or fragments thereof are
amplified by PCR and subcloned into the AdEasy shuttle vector
(Stratagene). Recombination and virus packaging are performed
according to the manufacturer's instructions to generate adenoviral
vectors. Alternatively, 158P1H4 coding sequences or fragments
thereof are cloned into the HSV-1 vector (Imgenex) to generate
herpes viral vectors. The viral vectors are thereafter used for
infection of various cell lines such as SCaBER, NIH 3T3, 293 or
rat-1 cells. The following regions of 158P1H4 are expressed in
these constructs, amino acids 1 to 440; or any 8, 9, 10, 11, 12,
13, 14, 15, or more contiguous amino acids from 158P1H4, variants,
or analogs thereof.
[0461] Regulated ExPression Systems: To control expression of
158P1H4 in mammalian cells, coding sequences of 158P1H4 are cloned
into regulated mammalian expression systems such as the T-Rex
System (Invitrogen), the GeneSwitch System (Invitrogen) and the
tightly-regulated Ecdysone System (Sratagene). These systems allow
the study of the temporal and concentration dependent effects of
recombinant 158P1H4. These vectors are thereafter used to control
expression of 158P1H4 in various cell lines such as SCaBER, NIH
3T3, 293 or rat1 cells. The following regions of 158P1H4 are
expressed in these constructs, amino acids I to 440; or any 8, 9,
10, 11, 12, 13, 14, 15, or more contiguous amino acids from
158P1H4, variants, or analogs thereof.
[0462] B. Baculovirus Expression Systems
[0463] To generate recombinant 158P1H4 proteins in a baculovirus
expression system, 158P1H4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1H4 are cloned into the baculovirus
transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag
at the N-terminus. Specifically, pBlueBac-158P1H4 is co-transfected
with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera
frugiperda) insect cells to generate recombinant baculovirus (see
Invitrogen instruction manual for details). Baculovirus is then
collected from cell supernatant and purified by plaque assay.
[0464] Recombinant 158P1H4 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 158P1H4 protein can be detected using anti-158P1H4 or
anti-His-tag antibody. 158P1H4 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 158P1H4.
[0465] The following regions of 158P1H4 are expressed in these
constructs, amino acids 1 to 440; or any 8, 9, 10, 11, 12, 13, 14,
15, or more contiguous amino acids from 158P1H4, variants, or
analogs thereof.
Example 7
[0466] Antigenicity Profiles
[0467] FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13 depict
graphically five amino acid profiles of the 158P1H4 amino acid
sequence, each assessment available by accessing the ProtScale
website (URL www.expasy.ch/cgi-bin/protscale.pl) on the ExPasy
molecular biology server.
[0468] These profiles: FIG. 9, Hydrophilicity, (Hopp T. P., Woods
K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 10,
Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.
157:105-132); FIG. 11, Percentage Accessible Residues (Janin J.,
1979 Nature 277:491-492); FIG. 12, Average Flexibility, (Bhaskaran
R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.
32:242-255); FIG. 13, Beta-turn (Deleage, G., Roux B. 1987 Protein
Engineering 1:289-294); and optionally others available in the art,
such as on the ProtScale website, were used to identify antigenic
regions of the 158P1H4 protein. Each of the above amino acid
profiles of 158P1H4 were generated using the following ProtScale
parameters for analysis: 1) A window size of 9; 2) 100% weight of
the window edges compared to the window center; and, 3) amino acid
profile values normalized to lie between 0 and 1.
[0469] Hydrophilicity (FIG. 9), Hydropathicity (FIG. 10) and
Percentage Accessible Residues (FIG. 11) profiles were used to
determine stretches of hydrophilic amino acids (i.e., values
greater than 0.5 on the Hydrophilicity and Percentage Accessible
Residues profile, and values less than 0.5 on the Hydropathicity
profile). Such regions are likely to be exposed to the aqueous
environment, be present on the surface of the protein, and thus
available for immune recognition, such as by antibodies.
[0470] Average Flexibility (FIG. 12) and Beta-turn (FIG. 13)
profiles determine stretches of amino acids (i.e., values greater
than 0.5 on the Beta-turn profile and the Average Flexibility
profile) that are not constrained in secondary structures such as
beta sheets and alpha helices. Such regions are also more likely to
be exposed on the protein and thus accessible to immune
recognition, such as by antibodies.
[0471] Antigenic sequences of the 158P1H4 protein indicated, e.g.,
by the profiles set forth in FIG. 9, FIG. 10, FIG. 11, FIG. 12, or
FIG. 13 are used to prepare immunogens, either peptides or nucleic
acids that encode them, to generate therapeutic and diagnostic
anti-158P1H4 antibodies. The immunogen can be any 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 25, 30,
35, 40, 45, 50 or more than 50 contiguous amino acids, or the
corresponding nucleic acids that encode them, from the 158P1H4
protein. In particular, peptide immunogens of the invention can
comprise, a peptide region of at least 5 amino acids of FIG. 2 in
any whole number increment up to 440 that includes an amino acid
position having a value greater than 0.5 in the Hydrophilicity
profile of FIG. 9; a peptide region of at least 5 amino acids of
FIG. 2 in any whole number increment up to 440 that includes an
amino acid position having a value less than 0.5 in the
Hydropathicity profile of FIG. 10; a peptide region of at least 5
amino acids of FIG. 2 in any whole number increment up to 440 that
includes an amino acid position having a value greater than 0.5 in
the Percent Accessible Residues profile of FIG. 11; a peptide
region of at least 5 amino acids of FIG. 2 in any whole number
increment up to 440 that includes an amino acid position having a
value greater than 0.5 in the Average Flexibility profile on FIG.
12; and, a peptide region of at least 5 amino acids of FIG. 2 in
any whole number increment up to 440 that includes an amino acid
position having a value greater than 0.5 in the Beta-turn profile
of FIG. 13. Peptide immunogens of the invention can also comprise
nucleic acids that encode any of the forgoing. All immunogens of
the invention, peptide or nucleic acid, can be embodied in human
unit dose form, or comprised by a composition that includes a
pharmaceutical excipient compatible with human physiology.
Example 8
[0472] Generation of 158P1H4 Polyclonal Antibodies
[0473] Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. In addition to immunizing with the full
length 158P1H4 protein, computer algorithms are employed in design
of immunogens that, based on amino acid sequence analysis contain
characteristics of being antigenic and available for recognition by
the immune system of the immunized host (see the Example entitled
"Antigenicity Profiles"). Such regions would be predicted to be
hydrophilic, flexible, in beta-turn conformations, and be exposed
on the surface of the protein (see, e.g., FIG. 9, FIG. 10, FIG. 11,
FIG. 12, or FIG. 13 for amino acid profiles that indicate such
regions of 158P1H4).
[0474] For example, 158P1H4 recombinant bacterial fusion proteins
or peptides encoding hydrophilic, flexible, beta-turn regions of
the 158P1H4 sequence, such as amino acids 10-22 and 370440 are used
as antigens to generate polyclonal antibodies in New Zealand White
rabbits. It is useful to conjugate the immunizing agent to a
protein known to be immunogenic in the mammal being immunized.
Examples of such immunogenic proteins include, but are not limited
to, keyhole limpet hemocyanin (KLH), serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a
peptide encoding amino acids 10-22 of 158P1H4 is conjugated to KLH
and used to immunize the rabbit. Alternatively the immunizing agent
may include all or portions of the 158P1H4 protein, analogs or
fusion proteins thereof. For example, the 158P1H4 amino acid
sequence can be fused using recombinant DNA techniques to any one
of a variety of fusion protein partners that are well known in the
art, such as glutathione-S-transferase (GST) and HIS tagged fusion
proteins. Such fusion proteins are purified from induced bacteria
using the appropriate affinity matrix. In one embodiment, a
GST-fusion protein encoding amino acids 370-440 of 158P1H4 is
produced and purified, and a cleavage product is generated in which
GST sequences are removed by proteolytic cleavage. This cleaved
158P1H4 protein is used as immunogen. Other recombinant bacterial
fusion proteins that may be employed include maltose binding
protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant
region (see the section entitled "Production of 158P1H4 in
Prokaryotic Systems" and Current Protocols In Molecular Biology,
Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley,
P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and
Ledbetter, L.(1991) J.Exp. Med. 174, 561-566).
[0475] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see the section entitled "Production of
Recombinant 158P1H4 in Eukaryotic Systems"), and retain
post-translational modifications such as glycosylations found in
native 158P1H4 protein. In one embodiment, a predicted antigenic
region of 158P1H4, amino acids 370-440, is cloned into the Tag5
mammalian secretion vector. The recombinant protein is purified by
metal chelate chromatography from tissue culture supernatants of
293T cells stably expressing the recombinant vector. The purified
Tag5 158P1H4 protein is then used as immunogen.
[0476] During the immunization protocol, it is useful to mix or
emulsify the antigen in adjuvants that enhance the immune response
of the host animal. Examples of adjuvants include, but are not
limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
[0477] In a typical protocol, rabbits are initially immunized
subcutaneously with up to 200 .mu.g, typically 100-200 .mu.g, of
fusion protein or peptide conjugated to KLH mixed in complete
Freund's adjuvant (CFA). Rabbits are then injected subcutaneously
every two weeks with up to 200 .mu.g, typically 100-200 .mu.g, of
the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds
are taken approximately 7-10 days following each immunization and
used to monitor the titer of the antiserum by ELISA.
[0478] To test serum, such as rabbit serum, for reactivity with
158P1H4 proteins, the full-length 158P1H4 cDNA can be cloned into
an expression vector such as one that provides a 6.times.His tag at
the carboxyl-terminus (pCDNA 3.1 myc-his, Invitrogen, see the
Example entitled "Production of Recombinant 158P1H4 in Eukaryotic
Systems"). After transfection of the constructs into 293T cells,
cell lysates are probed with the anti-158P1H4 serum and with
anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.)
to determine specific reactivity to denatured 158P1H4 protein using
the Western blot technique. In addition, recognition of native
protein by the antiserum can be determined by immunoprecipitation
and flow cytometric analyses of 293T and other recombinant
158P1H4-expressing cells. Alternatively, specificity of the
antiserum is tested by Western blot, immunoprecipitation,
fluorescent microscopy, and flow cytometric techniques using cells
that endogenously express 158P1H4.
[0479] Sera from rabbits immunized with fusion proteins, such as
GST and MBP fusion proteins, are purified by depletion of
antibodies reactive to GST, MBP, or other fusion partner sequence
by passage over an affinity column containing the fusion partner
either alone or in the context of an irrelevant fusion protein.
Sera from His-tagged protein and peptide immunized rabbits as well
as fusion partner depleted sera are further purified by passage
over an affinity column composed of the original protein immunogen
or free peptide coupled to Affigel matrix (BioRad).
Example 9
[0480] Generation of 158P1H4 Monoclonal Antibodies (mAbs)
[0481] In one embodiment, therapeutic mAbs to 158P1H4 comprise
those that react with epitopes of the protein that would disrupt or
modulate the biological function of 158P1H4, for example those that
would disrupt its interaction with ligands or proteins that mediate
or are involved in its biological activity. Immunogens for
generation of such mAbs include those designed to encode or contain
the entire 158P1H4 protein or regions of the 158P1H4 protein
predicted to be antigenic from computer analysis of the amino acid
sequence (see, e.g., FIG. 9, FIG. 10, FIG. 11, FIG. 12, or FIG. 13,
and the Example entitled "Antigenicity Profiles").
[0482] Immunogens include peptides, recombinant bacterial proteins,
and mammalian expressed Tag 5 proteins and human and murine IgG FC
fusion proteins. To generate mabs to 158P1H4, mice are first
immunized intraperitoneally (IP) with, typically, 10-50 .mu.g of
protein immunogen mixed in complete Freund's adjuvant. Mice are
then subsequently immunized IP every 2-4 weeks with, typically,
10-50 .mu.g of antigen mixed in incomplete Freund's adjuvant.
Alternatively, MPL-TDM adjuvant is used in immunizations. In
addition, a DNA-based immunization protocol is employed in which a
mammalian expression vector encoding 158P1H4 sequence is used to
immunize mice by direct injection of the plasmid DNA. For example,
either pCDNA 3.1 encoding the full length 158P1H4 cDNA, or amino
acids 370-440 of 158P1H4 (predicted to be antigenic from sequence
analysis, see, e.g., FIG. 9, FIG. 10, FIG. 11, FIG. 12 or FIG. 13)
fused at the amino-terminus to an IgK leader sequence and at the
carboxyl-terminus to the coding sequence of murine or human IgG Fc
region, is used. This protocol is used alone and in combination
with protein immunogens. Test bleeds are taken 7-10 days following
immunization to monitor titer and specificity of the immune
response. Once appropriate reactivity and specificity is obtained
as determined by ELISA, Western blotting, immunoprecipitation,
fluorescence microscopy, and flow cytometric analyses, fusion and
hybridoma generation is then carried out with established
procedures well known in the art (see, e.g., Harlow and Lane,
1988).
[0483] In one embodiment for generating 158P1H4 monoclonal
antibodies, a glutathione-S-transferase (GST) fusion protein
encoding amino acids 370-440 of 158P1H4 protein is expressed and
purified. A cleavage fragment encoding 158P1H4 specific amino acids
is then used as immunogen in which GST is removed by site-specific
proteolysis. Balb C mice are initially immunized intraperitoneally
with 25 .mu.g of the 158P1H4 cleavage protein mixed in complete
Freund's adjuvant. Mice are subsequently immunized every two weeks
with 25 .mu.g of 158P1H4 cleavage protein mixed in incomplete
Freund's adjuvant for a total of three immunizations. The titer of
serum from immunized mice is determined by ELISA using the full
length GST-fusion protein and the cleaved immunogen. Reactivity and
specificity of serum to full length 158P1H4 protein is monitored by
Western blotting, immunoprecipitation and flow cytometry using 293T
cells transfected with an expression vector encoding the 158P1H4
cDNA (see e.g., the Example entitled "Production of Recombinant
158P1H4 in Bacterial and Mammalian Systems"). Other recombinant
158P1H4-expressing cells or cells endogenously expressing 158P1H4
are also used. Mice showing the strongest reactivity are rested and
given a final injection of 158P1H4 cleavage protein in PBS and then
sacrificed four days later. The spleens of the sacrificed mice are
harvested and fused to SPO/2 myeloma cells using standard
procedures (Harlow and Lane, 1988). Supernatants from growth wells
following HAT selection are screened by ELISA, Western blot,
immunoprecipitation, fluorescent microscopy, and flow cytometry to
identify 158P1H4 specific antibody-producing clones.
[0484] The binding affinity of a 158P1H4 monoclonal antibody is
determined using standard technologies. Affinity measurements
quantify the strength of antibody to epitope binding and are used
to help define which 158P1H4 monoclonal antibodies preferred for
diagnostic or therapeutic use, as appreciated by one of skill in
the art. The BIAcore system (Uppsala, Sweden) is a preferred method
for determining binding affinity. The BIAcore system uses surface
plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1;
Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor
biomolecular interactions in real time. BIAcore analysis
conveniently generates association rate constants, dissociation
rate constants, equilibrium dissociation constants, and affinity
constants.
Example 10
[0485] HLA Class I and Class II Binding Assays
[0486] HLA class I and class II binding assays using purified HLA
molecules are performed in accordance with disclosed protocols
(e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J.
Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813
(1994)). Briefly, purified MHC molecules (5 to 500 nM) are
incubated with various unlabeled peptide inhibitors and 1-10 nM
.sup.125I-radiolabeled probe peptides as described. Following
incubation, MHC-peptide complexes are separated from free peptide
by gel filtration and the fraction of peptide bound is determined.
Typically, in preliminary experiments, each MHC preparation is
titered in the presence of fixed amounts of radiolabeled peptides
to determine the concentration of HLA molecules necessary to bind
10-20% of the total radioactivity. All subsequent inhibition and
direct binding assays are performed using these HLA
concentrations.
[0487] Since under these conditions [label]<[HLA] and
IC.sub.50.gtoreq.[IILA], the measured IC.sub.50 values are
reasonable approximations of the true K.sub.D values. Peptide
inhibitors are typically tested at concentrations ranging from 120
.mu.g/ml to 1.2 ng/ml, and are tested in two to four completely
independent experiments. To allow comparison of the data obtained
in different experiments, a relative binding figure is calculated
for each peptide by dividing the IC.sub.50 of a positive control
for inhibition by the IC.sub.50 for each tested peptide (typically
unlabeled versions of the radiolabeled probe peptide). For database
purposes, and inter-experiment comparisons, relative binding values
are compiled. These values can subsequently be converted back into
IC.sub.50 nM values by dividing the IC.sub.50 nM of the positive
controls for inhibition by the relative binding of the peptide of
interest. This method of data compilation is accurate and
consistent for comparing peptides that have been tested on
different days, or with different lots of purified MHC.
[0488] Binding assays as outlined above may be used to analyze HLA
supermotif and/or HLA motif-bearing peptides.
Example 11
[0489] Identification of HLA Supermotif- and Motif-Bearing CTL
Candidate Epitopes
[0490] HLA vaccine compositions of the invention can include
multiple epitopes. The multiple epitopes can comprise multiple HLA
supermotifs or motifs to achieve broad population coverage. This
example illustrates the identification and confirmation of
supermotif- and motif-bearing epitopes for the inclusion in such a
vaccine composition. Calculation of population coverage is
performed using the strategy described below.
[0491] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-bearing Epitopes
[0492] The searches performed to identify the motif-bearing peptide
sequences in the Example entitled "Antigenicity Profiles" and
Tables V-XVIII employ the protein sequence data from the gene
product of 158P1H4 set forth in FIGS. 2 and 3.
[0493] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
158P1H4 protein sequences are analyzed using a text string search
software program to identify potential peptide sequences containing
appropriate HLA binding motifs; such programs are readily produced
in accordance with information in the art in view of known
motif/supermotif disclosures. Furthermore, such calculations can be
made mentally.
[0494] Identified A2-, A3-, and DR-supermotif sequences are scored
using polynomial algorithms to predict their capacity to bind to
specific HLA-Class I or Class II molecules. These polynomial
algorithms account for the impact of different amino acids at
different positions, and are essentially based on the premise that
the overall affinity (or .DELTA.G) of peptide-HLA molecule
interactions can be approximated as a linear polynomial function of
the type:
".DELTA.G"=a.sub.1ix a.sub.2ix a.sub.3i . . . x a.sub.ni
[0495] where a.sub.j1 is a coefficient which represents the effect
of the presence of a given amino acid (j) at a given position (i)
along the sequence of a peptide of n amino acids. The crucial
assumption of this method is that the effects at each position are
essentially independent of each other (i.e., independent binding of
individual side-chains). When residue j occurs at position i in the
peptide, it is assumed to contribute a constant amount j.sub.i to
the free energy of binding of the peptide irrespective of the
sequence of the rest of the peptide.
[0496] The method of derivation of specific algorithm coefficients
has been described in Gulukota et al., J. Mol. Biol. 267:1258-126,
1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and
Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for
all i positions, anchor and non-anchor alike, the geometric mean of
the average relative binding (ARB) of all peptides carrying j is
calculated relative to the remainder of the group, and used as the
estimate of j.sub.i. For Class II peptides, if multiple alignments
are possible, only the highest scoring alignment is utilized,
following an iterative procedure. To calculate an algorithm score
of a given peptide in a test set, the ARB values corresponding to
the sequence of the peptide are multiplied. If this product exceeds
a chosen threshold, the peptide is predicted to bind. Appropriate
thresholds are chosen as a function of the degree of stringency of
prediction desired.
[0497] Selection of HLA-A2 Supertype Cross-reactive Peptides
[0498] Complete protein sequences from 158P1H4 are scanned
utilizing motif identification software, to identify 8-, 9- 10- and
11 -mer sequences containing the HLA-A2-supermotif main anchor
specificity. Typically, these sequences are then scored using the
protocol described above and the peptides corresponding to the
positive-scoring sequences are synthesized and tested for their
capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201
is considered a prototype A2 supertype molecule).
[0499] These peptides are then tested for the capacity to bind to
additional A2-supertype molecules (A*0202, A*0203, A*0206, and
A*6802). Peptides that bind to at least three of the five
A2-supertype alleles tested are typically deemed A2-supertype
cross-reactive binders. Preferred peptides bind at an affinity
equal to or less than 500 nM to three or more HLA-A2 supertype
molecules.
[0500] Selection of HLA-A3 Supermotif-bearing Epitopes
[0501] The 158P1H4 protein sequence scanned above is also examined
for the presence of peptides with the HLA-A3-supermotif primary
anchors. Peptides corresponding to the HLA A3 supermotif-bearing
sequences are then synthesized and tested for binding to HLA-A*0301
and HLA-A*1101 molecules, the molecules encoded by the two most
prevalent A3-supertype alleles. The peptides that bind at least one
of the two alleles with binding affinities of .ltoreq.500 nM, often
.ltoreq.200 nM, are then tested for binding cross-reactivity to the
other common A3-supertype alleles (e.g., A*3101, A*3301, and
A*6801) to identify those that can bind at least three of the five
HLA-A3-supertype molecules tested.
[0502] Selection of HLA-B7 Supermotif Bearing Epitopes
[0503] The 158P1H4 protein is also analyzed for the presence of 8-,
9- 10-, or 1 1-mer peptides with the HLA-B7-supermotif.
Corresponding peptides are synthesized and tested for binding to
HLA-B*0702, the molecule encoded by the most common B7-supertype
allele (i.e., the prototype B7 supertype allele). Peptides binding
B*0702 with IC.sub.50 of .ltoreq.500 nM are identified using
standard methods. These peptides are then tested for binding to
other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301,
and B*5401). Peptides capable of binding to three or more of the
five B7-supertype alleles tested are thereby identified.
[0504] Selection of A1 and A24 Motif-bearing Epitopes
[0505] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 158P1H4 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0506] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs are identified using analogous
methodology.
Example 12
[0507] Confirmation of Immunogenicity
[0508] Cross-reactive candidate CTL A2-supermotif-bearing peptides
that are identified as described herein are selected to confirm in
vitro immunogenicity. Confirmation is performed using the following
methodology:
[0509] Target Cell Lines for Cellular Screening
[0510] The .221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of HLA-A2.1-restricted CTL. This cell
line is grown in RPMI-1640 medium supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (v/v) heat
inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the antigen of interest,
can be used as target cells to confirm the ability of
peptide-specific CTLs to recognize endogenous antigen.
[0511] Primary CTL Induction Cultures
[0512] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI
with 30 .mu.g/ml DNAse, washed twice and resuspended in complete
medium (RPMI-1640 plus 5% AB human serum, non-essential amino
acids, sodium pyruvate, L-glutamine and penicillin/streptomycin).
The monocytes are purified by plating 10.times.10.sup.6 PBMC/well
in a 6-well plate. After 2 hours at 37.degree. C., the non-adherent
cells are removed by gently shaking the plates and aspirating the
supernatants. The wells are washed a total of three times with 3 ml
RPMI to remove most of the non-adherent and loosely adherent cells.
Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000
U/ml of IL-4 are then added to each well. TNF.alpha. is added to
the DCs on day 6 at 75 ng/ml and the cells are used for CTL
induction cultures on day 7.
[0513] Induction of CTL with DC and Peptide: CD8+ T-cells are
isolated by positive selection with Dynal immunomagnetic beads
(Dynabeads.RTM. M-450) and the detacha-bead.RTM. reagent. Typically
about 200-250.times.10.sup.6 PBMC are processed to obtain
24.times.10.sup.6 CD8.sup.+ T-cells (enough for a 48-well plate
culture). Briefly, the PBMCs are thawed in RPMI with 30 .mu.g/ml
DNAse, washed once with PBS containing 1% human AB serum and
resuspended in PBS/1% AB serum at a concentration of
20.times.10.sup.6cells/ml. The magnetic beads are washed 3 times
with PBS/AB serum, added to the cells (140 .mu.l
beads/20.times.10.sup.6 cells) and incubated for 1 hour at
4.degree. C. with continuous mixing. The beads and cells are washed
4.times. with PBS/AB serum to remove the nonadherent cells and
resuspended at 100.times.10.sup.6 cells/ml (based on the original
cell number) in PBS/AB serum containing 100 .mu.l/ml
detacha-bead.RTM. reagent and 30 82 g/ml DNAse. The mixture is
incubated for 1 hour at room temperature with continuous mixing.
The beads are washed again with PBS/AB/DNAse to collect the CD8+
T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7
minutes, washed once with PBS with 1% BSA, counted and pulsed with
40 .mu.g/ml of peptide at a cell concentration of
1-2.times.10.sup.6/ml in the presence of 3 .mu.g/ml .beta..sub.2-
microglobulin for 4 hours at 20.degree. C. The DC are then
irradiated (4,200 rads), washed 1 time with medium and counted
again.
[0514] Setting up induction cultures: 0.25 ml cytokine-generated DC
(at 1.times.10.sup.5 cells/ml) are co-cultured with 0.25 ml of CD8+
T-cells (at 2.times.10.sup.6 cell/ml) in each well of a 48-well
plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10
is added the next day at a final concentration of 10 ng/ml and
human IL-2 is added 48 hours later at 10 IU/ml.
[0515] Restimulation of the induction cultures with peptide-pulsed
adherent cells: Seven and fourteen days after the primary
induction, the cells are restimulated with peptide-pulsed adherent
cells. The PBMCs are thawed and washed twice with RPMI and DNAse.
The cells are resuspended at 5.times.10.sup.6 cells/ml and
irradiated at .about.4200 rads. The PBMCs are plated at
2.times.10.sup.6 in 0.5 ml complete medium per well and incubated
for 2 hours at 37.degree. C. The plates are washed twice with RPMI
by tapping the plate gently to remove the nonadherent cells and the
adherent cells pulsed with 10 .mu.g/ml of peptide in the presence
of 3 .mu.g/ml .beta..sub.2 microglobulin in 0.25 ml RPMI/5%AB per
well for 2 hours at 37.degree. C. Peptide solution from each well
is aspirated and the wells are washed once with RPMI. Most of the
media is aspirated from the induction cultures (CD8+ cells) and
brought to 0.5 ml with fresh media. The cells are then transferred
to the wells containing the peptide-pulsed adherent cells. Twenty
four hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant human IL2 is added the
next day and again 2-3 days later at 50IU/ml (Tsai et al, Critical
Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the
cultures are assayed for CTL activity in a .sup.51Cr release assay.
In some experiments the cultures are assayed for peptide-specific
recognition in the in situ IFN.gamma. ELISA at the time of the
second restimulation followed by assay of endogenous recognition 7
days later. After expansion, activity is measured in both assays
for a side-by-side comparison.
[0516] Measurement of CTL Lytic Activity by .sup.51Cr Release
[0517] Seven days after the second restimulation, cytotoxicity is
determined in a standard (5 hr) .sup.51Cr release assay by assaying
individual wells at a single E:T. Peptide-pulsed targets are
prepared by incubating the cells with 10 .mu.g/ml peptide overnight
at 37.degree. C.
[0518] Adherent target cells are removed from culture flasks with
trypsin-EDTA. Target cells are labelled with 200 .mu.Ci of
.sup.51Cr sodium chromate (Dupont, Wilmington, DE) for 1 hour at
37.degree. C. Labelled target cells are resuspended at 10.sup.6 per
ml and diluted 1:10 with K562 cells at a concentration of
3.3.times.10.sup.6/ml (an NK-sensitive erytroblastoma cell line
used to reduce non-specific lysis). Target cells (100 .mu.l) and
effectors (100 .mu.l) are plated in 96 well round-bottom plates and
incubated for 5 hours at 37.degree. C. At that time, 100 .mu.l of
supernatant are collected from each well and percent lysis is
determined according to the formula: [(cpm of the test sample- cpm
of the spontaneous .sup.51Cr release sample)/(cpm of the maximal
.sup.51Cr release sample- cpm of the spontaneous .sup.51Cr release
sample)].times.100.
[0519] Maximum and spontaneous release are determined by incubating
the labelled targets with 1% Trition X-100 and media alone,
respectively. A positive culture is defined as one in which the
specific lysis (sample- background) is 10% or higher in the case of
individual wells and is 15% or more at the two highest E:T ratios
when expanded cultures are assayed.
[0520] In situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-specific and Endogenous Recognition
[0521] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH8.2) overnight
at 4.degree. C. The plates are washed with Ca.sup.2+,
Mg.sup.2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for
two hours, after which the CTLs (100 .mu.l/well) and targets (100
.mu./well) are added to each well, leaving empty wells for the
standards and blanks (which received media only). The target cells,
either peptide-pulsed or endogenous targets, are used at a
concentration of 1.times.10.sup.6 cells/ml. The plates are
incubated for 48 hours at 37.degree. C. with 5% CO.sub.2.
[0522] Recombinant human IFN-gamma is added to the standard wells
starting at 400 pg or 1200 pg/100 microliter/well and the plate
incubated for two hours at 37.degree. C. The plates are washed and
100 .mu.l of biotinylated mouse anti-human IFN-gamma monoclonal
antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and
incubated for 2 hours at room temperature. After washing again, 100
microliter HRP-streptavidin (1:4000) are added and the plates
incubated for one hour at room temperature. The plates are then
washed 6.times. with wash buffer, 100 microliter/well developing
solution (TMB 1:1) are added, and the plates allowed to develop for
5-15 minutes. The reaction is stopped with 50 microliter/well 1M
H.sub.3PO.sub.4 and read at OD450. A culture is considered positive
if it measured at least 50 pg of IFN-gamma/well above background
and is twice the background level of expression.
[0523] CTL Expansion
[0524] Those cultures that demonstrate specific lytic activity
against peptide-pulsed targets and/or tumor targets are expanded
over a two week period with anti-CD3. Briefly, 5.times.10.sup.4
CD8+ cells are added to a T25 flask containing the following:
1.times.10.sup.6 irradiated (4,200 rad) PBMC (autologous or
allogeneic) per ml, 2.times.10.sup.5 irradiated (8,000 rad)
EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml
in RPMI-1640 containing 10% (v/v) human AB serum, non-essential
amino acids, sodium pyruvate, 25 .mu.M 2-mercaptoethanol,
L-glutamine and penicillin/streptomycin. Recombinant human IL2 is
added 24 hours later at a final concentration of 200 IU/ml and
every three days thereafter with fresh media at 50 IU/ml. The cells
are split if the cell concentration exceeds 1.times.10.sup.6/ml and
the cultures are assayed between days 13 and 15 at E:T ratios of
30, 10, 3 and 1:1 in the .sup.51Cr release assay or at
1.times.10.sup.6/ml in the in situ IFN.gamma. assay using the same
targets as before the expansion.
[0525] Cultures are expanded in the absence of anti-CD3' as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8.sup.+ cells are added to a T25 flask
containing the following: 1.times.10.sup.6 autologous PBMC per ml
which have been peptide-pulsed with 10 .mu.g/ml peptide for two
hours at 37.degree. C. and irradiated (4,200 rad); 2.times.10.sup.5
irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640
containing 10%(v/v) human AB serum, non-essential AA, sodium
pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0526] Immunogenicity of A2 Supermotif-bearing Peptides
[0527] A2-supermotif cross-reactive binding peptides are tested in
the cellular assay for the ability to induce peptide-specific CTL
in normal individuals. In this analysis, a peptide is typically
considered to be an epitope if it induces peptide-specific CTLs in
at least individuals, and preferably, also recognizes the
endogenously expressed peptide.
[0528] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 158P1H4. Briefly,
PBMCs are isolated from patients, re-stimulated with peptide-pulsed
monocytes and assayed for the ability to recognize peptide-pulsed
target cells as well as transfected cells endogenously expressing
the antigen.
[0529] Evaluation of A*03/A11 Immunogenicity
[0530] HLA-A3 supermotif-bearing cross-reactive binding peptides
are also evaluated for immunogenicity using methodology analogous
for that used to evaluate the immunogenicity of the HLA-A2
supermotif peptides.
[0531] Evaluation of B7 Inmunogenicity
[0532] Immunogenicity screening of the B7-supertype cross-reactive
binding peptides identified as set forth herein are confirmed in a
manner analogous to the confirmation of A2- and
A3-supermotif-bearing peptides.
[0533] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also confirmed using similar methodology
Example 13
[0534] Implementation of the Extended Supermotif to Improve the
Binding Capacity of Native Epitopes by CreatinE Analogs
[0535] HLA motifs and supermotifs (comprising primary and/or
secondary residues) are useful in the identification and
preparation of highly cross-reactive native peptides, as
demonstrated herein. Moreover, the definition of HLA motifs and
supermotifs also allows one to engineer highly cross-reactive
epitopes by identifying residues within a native peptide sequence
which can be analoged to confer upon the peptide certain
characteristics, e.g. greater cross-reactivity within the group of
HLA molecules that comprise a supertype, and/or greater binding
affinity for some or all of those HLA molecules. Examples of
analoging peptides to exhibit modulated binding affinity are set
forth in this example.
[0536] Analoging at Primary Anchor Residues
[0537] Peptide engineering strategies are implemented to further
increase the cross-reactivity of the epitopes. For example, the
main anchors of A2-supermotif-bearing peptides are altered, for
example, to introduce a preferred L, I, V, or M at position 2, and
I or V at the C-terminus.
[0538] To analyze the cross-reactivity of the analog peptides, each
engineered analog is initially tested for binding to the prototype
A2 supertype allele A*0201, then, if A*0201 binding capacity is
maintained, for A2-supertype cross-reactivity.
[0539] Alternatively, a peptide is confirmed as binding one or all
supertype members and then analogued to modulate binding affinity
to any one (or more) of the supertype members to add population
coverage.
[0540] The selection of analogs for immunogenicity in a cellular
screening analysis is typically further restricted by the capacity
of the parent wild type (WT) peptide to bind at least weakly, i.e.,
bind at an IC.sub.50 of 5000 nM or less, to three of more A2
supertype alleles. The rationale for this requirement is that the
WT peptides must be present endogenously in sufficient quantity to
be biologically relevant. Analoged peptides have been shown to have
increased immunogenicity and cross-reactivity by T cells specific
for the parent epitope (see, e.g., Parkhurst et al., J. Immunol.
157:2539, 1996; and Pogue et al., Proc. Natl Acad. Sci. USA
92:8166, 1995).
[0541] In the cellular screening of these peptide analogs, it is
important to confirm that analog-specific CTLs are also able to
recognize the wild-type peptide and, when possible, target cells
that endogenously express the epitope.
[0542] Analoging of HLA-A3 and B7-supermotif-bearing Peptides
[0543] Analogs of HLA-A3 supermotif-bearing epitopes are generated
using strategies similar to those employed in analoging HLA-A2
supermotif-bearing peptides. For example, peptides binding to 3/5
of the A3-supertype molecules are engineered at primary anchor
residues to possess a preferred residue (V, S, M, or A) at position
2.
[0544] The analog peptides are then tested for the ability to bind
A*03 and A*11 (prototype A3 supertype alleles). Those peptides that
demonstrate .ltoreq.500 nM binding capacity are then confirmed as
having A3-supertype cross-reactivity.
[0545] Similarly to the A2- and A3-motif bearing peptides, peptides
binding 3 or more B7-supertype alleles can be improved, where
possible, to achieve increased cross-reactive binding or greater
binding affinity or binding half life. B7 supermotif-bearing
peptides are, for example, engineered to possess a preferred
residue (V, I, L, or F) at the C-terminal primary anchor position,
as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490,
1996).
[0546] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0547] The analog peptides are then be confirmed for
immunogenicity, typically in a cellular screening assay. Again, it
is generally important to demonstrate that analog-specific CTLs are
also able to recognize the wild-type peptide and, when possible,
targets that endogenously express the epitope.
[0548] Analoging at Secondary Anchor Residues
[0549] Moreover, HLA supermotifs are of value in engineering highly
cross-reactive peptides and/or peptides that bind HLA molecules
with increased affinity by identifying particular residues at
secondary anchor positions that are associated with such
properties. For example, the binding capacity of a B7
supermotif-bearing peptide with an F residue at position 1 is
analyzed. The peptide is then analoged to, for example, substitute
L for F at position 1. The analoged peptide is evaluated for
increased binding affinity, binding half life and/or increased
cross-reactivity. Such a procedure identifies analoged peptides
with enhanced properties.
[0550] Engineered analogs with sufficiently improved binding
capacity or cross-reactivity can also be tested for immunogenicity
in HLA-B7-transgenic mice, following for example, IFA immunization
or lipopeptide immunization. Analogued peptides are additionally
tested for the ability to stimulate a recall response using PBMC
from patients with 158P1H4-expressing tumors.
[0551] Other Analoguing Strategies
[0552] Another form of peptide analoguing, unrelated to anchor
positions, involves the substitution of a cysteine with
.alpha.-amino butyric acid. Due to its chemical nature, cysteine
has the propensity to form disulfide bridges and sufficiently alter
the peptide structurally so as to reduce binding capacity.
Substitution of .alpha.-amino butyric acid for cysteine not only
alleviates this problem, but has been shown to improve binding and
crossbinding capabilities in some instances (see, e.g., the review
by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and
I. Chen, John Wiley & Sons, England, 1999).
[0553] Thus, by the use of single amino acid substitutions, the
binding properties and/or cross-reactivity of peptide ligands for
HLA supertype molecules can be modulated.
Example 14
[0554] Identification and confirmation of 158P1H4-derived sequences
with HLA-DR Binding Motifs
[0555] Peptide epitopes bearing an HLA class II supermotif or motif
are identified and confirmed as outlined below using methodology
similar to that described for HLA Class I peptides.
[0556] Selection of HLA-DR-supermotif-bearing Epitopes
[0557] To identify 158P1H4-derived, HLA class II HTL epitopes, the
158P1H4 antigen is analyzed for the presence of sequences bearing
an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are
selected comprising a DR-supermotif, comprising a 9-mer core, and
three-residue N- and C-terminal flanking regions (15 amino acids
total).
[0558] Protocols for predicting peptide binding to DR molecules
have been developed (Southwood et al., J. Immunol. 160:3363-3373,
1998). These protocols, specific for individual DR molecules, allow
the scoring, and ranking, of 9-mer core regions. Each protocol not
only scores peptide sequences for the presence of DR-supermotif
primary anchors (i.e., at position 1 and position 6) within a 9-mer
core, but additionally evaluates sequences for the presence of
secondary anchors. Using allele-specific selection tables (see,
e.g., Southwood et al., ibid.), it has been found that these
protocols efficiently select peptide sequences with a high
probability of binding a particular DR molecule. Additionally, it
has been found that performing these protocols in tandem,
specifically those for DR1, DR4w4, and DR7, can efficiently select
DR cross-reactive peptides.
[0559] The 158P1H4-derived peptides identified above are tested for
their binding capacity for various common HLA-DR molecules. All
peptides are initially tested for binding to the DR molecules in
the primary panel: DR1, DR4w4, and DR7. Peptides binding at least
two of these three DR molecules are then tested for binding to
DR2w2 .beta.1, DR2w2 .beta.2, DR6w19, and DR9 molecules in
secondary assays. Finally, peptides binding at least two of the
four secondary panel DR molecules, and thus cumulatively at least
four of seven different DR molecules, are screened for binding to
DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides
binding at least seven of the ten DR molecules comprising the
primary, secondary, and tertiary screening assays are considered
cross-reactive DR binders. 158P1H4-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0560] Selection of DR3 Motif Peptides
[0561] Because HLA-DR3 is an allele that is prevalent in Caucasian,
Black, and Hispanic populations, DR3 binding capacity is a relevant
criterion in the selection of HTL epitopes. Thus, peptides shown to
be candidates may also be assayed for their DR3 binding capacity.
However, in view of the binding specificity of the DR3 motif,
peptides binding only to DR3 can also be considered as candidates
for inclusion in a vaccine formulation.
[0562] To efficiently identify peptides that bind DR3, target
158P1H4 antigens are analyzed for sequences carrying one of the two
DR3-specific binding motifs reported by Geluk et al. (J. Immunol.
152:5742-5748, 1994). The corresponding peptides are then
synthesized and confirmed as having the ability to bind DR3 with an
affinity of 1 .mu.M or better, i.e., less than 1 .mu.M. Peptides
are found that meet this binding criterion and qualify as HLA class
II high affinity binders.
[0563] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0564] Similarly to the case of HLA class I motif-bearing peptides,
the class II motif-bearing peptides are analoged to improve
affinity or cross-reactivity. For example, aspartic acid at
position 4 of the 9-mer core sequence is an optimal residue for DR3
binding, and substitution for that residue often improves DR 3
binding.
Example 15
[0565] Immunogenicity of 158P1H4-derived HTL Epitopes
[0566] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0567] Immunogenicity of HTL epitopes are confirmed in a manner
analogous to the determination of immunogenicity of CTL epitopes,
by assessing the ability to stimulate HTL responses and/or by using
appropriate transgenic mouse models. Immunogenicity is determined
by screening for: 1.) in vitro primary induction using normal PBMC
or 2.) recall responses from patients who have 158P1H4-expressing
tumors.
Example 16
[0568] Calculation of Phenotypic Frequencies of HLA-supertypes in
Various Ethnic Backgrounds to Determine Breadth of Population
Coverage
[0569] This example illustrates the assessment of the breadth of
population coverage of a vaccine composition comprised of multiple
epitopes comprising multiple supermotifs and/or motifs.
[0570] In order to analyze population coverage, gene frequencies of
HLA alleles are determined. Gene frequencies for each HLA allele
are calculated from antigen or allele frequencies utilizing the
binomial distribution formulae gf=1-(SQRT(1-af)) (see, e.g., Sidney
et al., Human Immunol. 45:79-93, 1996). To obtain overall
phenotypic frequencies, cumulative gene frequencies are calculated,
and the cumulative antigen frequencies derived by the use of the
inverse formula [af=1-(1-Cgf).sup.2].
[0571] Where frequency data is not available at the level of DNA
typing, correspondence to the serologically defined antigen
frequencies is assumed. To obtain total potential supertype
population coverage no linkage disequilibrium is assumed, and only
alleles confirmed to belong to each of the supertypes are included
(minimal estimates). Estimates of total potential coverage achieved
by inter-loci combinations are made by adding to the A coverage the
proportion of the non-A covered population that could be expected
to be covered by the B alleles considered (e.g., total=A+B*(1-A)).
Confirmed members of the A3-like supertype areA3, A11, A31, A*3301,
and A*6801. Although the A3-like supertype may also include A34,
A66, and A*7401, these alleles were not included in overall
frequency calculations. Likewise, confirmed members of the A2-like
supertype family are A*0201, A*0202, A*0203, A*0204, A*0205,
A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like
supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301,
B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially a B*1401
B*3504-06, B*4201, and B*5602).
[0572] Population coverage achieved by combining the A2-, A3- and
B7-supertypes is approximately 86% in five major ethnic groups.
Coverage may be extended by including peptides bearing the A1 and
A24 motifs. On average, A1 is present in 12% and A24 in 29% of the
population across five different major ethnic groups (Caucasian,
North American Black, Chinese, Japanese, and Hispanic). Together,
these alleles are represented with an average frequency of 39% in
these same ethnic populations. The total coverage across the major
ethnicities when A1 and A24 are combined with the coverage of the
A2-, A3- and B7-supertype alleles is >95%. An analagous approach
can be used to estimate population coverage achieved with
combinations of class II motif-bearing epitopes.
[0573] Immunogenicity studies in humans (e.g., Bertoni et al., J.
Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997;
and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that
highly cross-reactive binding peptides are almost always recognized
as epitopes. The use of highly cross-reactive binding peptides is
an important selection criterion in identifying candidate epitopes
for inclusion in a vaccine that is immunogenic in a diverse
population.
[0574] With a sufficient number of epitopes (as disclosed herein
and from the art), an average population coverage is predicted to
be greater than 95% in each of five major ethnic populations. The
game theory Monte Carlo simulation analysis, which is known in the
art (see e.g., Osborne, M.J. and Rubinstein, A. "A course in game
theory" MIT Press, 1994), can be used to estimate what percentage
of the individuals in a population comprised of the Caucasian,
North American Black, Japanese, Chinese, and Hispanic ethnic groups
would recognize the vaccine epitopes described herein. A preferred
percentage is 90%. A more preferred percentage is 95%.
Example 17
[0575] CTL Recognition of Endogenously Processed Antigens After
Priming
[0576] This example confirms that CTL induced by native or analoged
peptide epitopes identified and selected as described herein
recognize endogenously synthesized, i.e., native antigens.
[0577] Effector cells isolated from transgenic mice that are
immunized with peptide epitopes, for example HLA-A2
supermotif-bearing epitopes, are re-stimulated in vitro using
peptide-coated stimulator cells. Six days later, effector cells are
assayed for cytotoxicity and the cell lines that contain
peptide-specific cytotoxic activity are further re-stimulated. An
additional six days later, these cell lines are tested for
cytotoxic activity on .sup.51Cr labeled Jurkat-A2.1/K.sup.b target
cells in the absence or presence of peptide, and also tested on
.sup.51Cr labeled target cells bearing the endogenously synthesized
antigen, i.e. cells that are stably transfected with 158P1H4
expression vectors.
[0578] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
158P1H4 antigen. The choice of transgenic mouse model to be used
for such an analysis depends upon the epitope(s) that are being
evaluated. In addition to HLA-A*0201/K.sup.b transgenic mice,
several other transgenic mouse models including mice with human Al
l, which may also be used to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for
HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed, which may be used to evaluate HTL
epitopes.
Example 18
[0579] Activity of CTL-HTL Conjugated Epitomes in Transgenic
Mice
[0580] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 158P1H4-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 158P1H4-expressing
tumor. The peptide composition can comprise multiple CTL and/or HTL
epitopes. The epitopes are identified using methodology as
described herein. This example also illustrates that enhanced
immunogenicity can be achieved by inclusion of one or more HTL
epitopes in a CTL vaccine composition; such a peptide composition
can comprise an HTL epitope conjugated to a CTL epitope. The CTL
epitope can be one that binds to multiple HLA family members at an
affinity of 500 nM or less, or analogs of that epitope. The
peptides may be lipidated, if desired.
[0581] Immunization procedures: Immunization of transgenic mice is
performed as described (Alexander et al., J. Immunol.
159:4753-4761, 1997). For example, A2/K.sup.b mice, which are
transgenic for the human HLA A2.1 allele and are used to confirm
the immunogenicity of HLA-A*0201 motif- or HLA-A2
supermotif-bearing epitopes, and are primed subcutaneously (base of
the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant,
or if the peptide composition is a lipidated CTL/HTL conjugate, in
DMSO/saline, or if the peptide composition is a polypeptide, in PBS
or Incomplete Freund's Adjuvant. Seven days after priming,
splenocytes obtained from these animals are restimulated with
syngenic irradiated LPS-activated lymphoblasts coated with
peptide.
[0582] Cell lines: Target cells for peptide-specific cytotoxicity
assays are Jurkat cells transfected with the HLA-A2.1/K.sup.b
chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007,
1991)
[0583] In vitro CTL activation: One week after priming, spleen
cells (30.times.10.sup.6 cells/flask) are co-cultured at 37.degree.
C. with syngeneic, irradiated (3000 rads), peptide coated
lymphoblasts (10.times.10.sup.6 cells/flask) in 10 culture
medium/T25 flask. After six days, effector cells are harvested and
assayed for cytotoxic activity.
[0584] Assay for cytotoxic activity: Target cells (1.0 to
1.5.times.10.sup.6) are incubated at 37.degree. C. in the presence
of 200 .mu.l of .sup.5Cr. After 60 minutes, cells are washed three
times and resuspended in R10 medium. Peptide is added where
required at a concentration of 1 .mu.g/ml. For the assay, 10.sup.4
51Cr-labeled target cells are added to different concentrations of
effector cells (final volume of 200 .mu.l) in U-bottom 96-well
plates. After a six hour incubation period at 37.degree. C., a 0.1
ml aliquot of supernatant is removed from each well and
radioactivity is determined in a Micromedic automatic gamma
counter. The percent specific lysis is determined by the formula:
percent specific release =100.times.(experimental
release--spontaneous release)/(maximum release--spontaneous
release). To facilitate comparison between separate CTL assays run
under the same conditions, % .sup.51Cr release data is expressed as
lytic units/10.sup.6 cells. One lytic unit is arbitrarily defined
as the number of effector cells required to achieve 30% lysis of
10,000 target cells in a six hour .sup.51Cr release assay. To
obtain specific lytic units/10.sup.6, the lytic units/10.sup.6
obtained in the absence of peptide is subtracted from the lytic
units/10.sup.6 obtained in the presence of peptide. For example, if
30% .sup.51Cr release is obtained at the effector (E): target (T)
ratio of 50:1 (i.e., 5.times.10.sup.5 effector cells for 10,000
targets) in the absence of peptide and 5:1 (i.e., 5.times.10.sup.4
effector cells for 10,000 targets) in the presence of peptide, the
specific lytic units would be:
[(1/50,000)-(1/500,000)].times.10.sup.6=18 LU.
[0585] The results are analyzed to assess the magnitude of the CTL
responses of animals injected with the immunogenic CTL/HTL
conjugate vaccine preparation and are compared to the magnitude of
the CTL response achieved using, for example, CTL epitopes as
outlined above in the Example entitled "Confirmation of
Immunogenicity". Analyses similar to this may be performed to
confirm the immunogenicity of peptide conjugates containing
multiple CTL epitopes and/or multiple HTL epitopes. In accordance
with these procedures, it is found that a CTL response is induced,
and concomitantly that an HTL response is induced upon
administration of such compositions.
Example 19
[0586] Selection of CTL and HTL Epitopes for Inclusion in an
158P1H4-specific Vaccine
[0587] This example illustrates a procedure for selecting peptide
epitopes for vaccine compositions of the invention. The peptides in
the composition can be in the form of a nucleic acid sequence,
either single or one or more sequences (i.e., minigene) that
encodes peptide(s), or can be single and/or polyepitopic
peptides.
[0588] The following principles are utilized when selecting a
plurality of epitopes for inclusion in a vaccine composition. Each
of the following principles is balanced in order to make the
selection.
[0589] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 158P1H4 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 158P1H4. For example, if it has been observed
that patients who spontaneously clear 158P1H4 generate an immune
response to at least three (3) from 158P1H4 antigen, then three or
four (3-4) epitopes should be included for HLA class I. A similar
rationale is used to determine HLA class II epitopes.
[0590] Epitopes are often selected that have a binding affinity of
an IC.sub.50 of 500 nM or less for an HLA class I molecule, or for
class II, an IC.sub.50 of 1000 nM or less; or HLA Class I peptides
with high binding scores from the BIMAS web site, at URL
bimas.dcrt.nih.gov/.
[0591] In order to achieve broad coverage of the vaccine through
out a diverse population, sufficient supermotif bearing peptides,
or a sufficient array of allele-specific motif bearing peptides,
are selected to give broad population coverage. In one embodiment,
epitopes are selected to provide at least 80% population coverage.
A Monte Carlo analysis, a statistical evaluation known in the art,
can be employed to assess breadth, or redundancy, of population
coverage.
[0592] When creating polyepitopic compositions, or a minigene that
encodes same, it is typically desirable to generate the smallest
peptide possible that encompasses the epitopes of interest. The
principles employed are similar, if not the same, as those employed
when selecting a peptide comprising nested epitopes. For example, a
protein sequence for the vaccine composition is selected because it
has maximal number of epitopes contained within the sequence, ie.,
it has a high concentration of epitopes. Epitopes may be nested or
overlapping (i.e., frame shifted relative to one another). For
example, with overlapping epitopes, two 9-mer epitopes and one
10-mer epitope can be present in a 10 amino acid peptide. Each
epitope can be exposed and bound by an HLA molecule upon
administration of such a peptide. A multi-epitopic, peptide can be
generated synthetically, recombinantly, or via cleavage from the
native source. Alternatively, an analog can be made of this native
sequence, whereby one or more of the epitopes comprise
substitutions that alter the cross-reactivity and/or binding
affinity properties of the polyepitopic peptide. Such a vaccine
composition is administered for therapeutic or prophylactic
purposes. This embodiment provides for the possibility that an as
yet undiscovered aspect of immune system processing will apply to
the native nested sequence and thereby facilitate the production of
therapeutic or prophylactic immune response-inducing vaccine
compositions. Additionally such an embodiment provides for the
possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment (absent the
creating of any analogs) directs the immune response to multiple
peptide sequences that are actually present in 158P1H4, thus
avoiding the need to evaluate any junctional epitopes. Lastly, the
embodiment provides an economy of scale when producing nucleic acid
vaccine compositions. Related to this embodiment, computer programs
can be derived in accordance with principles in the art, which
identify in a target sequence, the greatest number of epitopes per
sequence length.
[0593] A vaccine composition comprised of selected peptides, when
administered, is safe, efficacious, and elicits an immune response
similar in magnitude to an immune response that controls or clears
cells that bear or overexpress 158P1H4.
Example 20
[0594] Construction of "Minigene" Multi-Epitope DNA Plasmids
[0595] This example discusses the construction of a minigene
expression plasmid. Minigene plasmids may, of course, contain
various configurations of B cell, CTL and/or HTL epitopes or
epitope analogs as described herein.
[0596] A minigene expression plasmid typically includes multiple
CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3,
-B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24
motif-bearing peptide epitopes are used in conjunction with DR
supermotif-bearing epitopes and/or DR3 epitopes. HLA class I
supermotif or motif-bearing peptide epitopes derived 158P1H4, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 158P1H4 to provide broad population coverage,
i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3
motif-bearing epitopes are selected for inclusion in the minigene
construct. The selected CTL and HTL epitopes are then incorporated
into a minigene for expression in an expression vector.
[0597] Such a construct may additionally include sequences that
direct the HTL epitopes to the endoplasmic reticulum. For example,
the Ii protein may be fused to one or more HTL epitopes as
described in the art, wherein the CLIP sequence of the Ii protein
is removed and replaced with an HLA class II epitope sequence so
that HLA class II epitope is directed to the endoplasmic reticulum,
where the epitope binds to an HLA class II molecules.
[0598] This example illustrates the methods to be used for
construction of a minigene-bearing expression plasmid. Other
expression vectors that may be used for minigene compositions are
available and known to those of skill in the art.
[0599] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa Ig-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disclosed herein. The sequence
encodes an open reading frame fused to the Myc and His antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0600] Overlapping oligonucleotides that can, for example, average
about 70 nucleotides in length with 15 nucleotide overlaps, are
synthesized and HPLC-purified. The oligonucleotides encode the
selected peptide epitopes as well as appropriate linker
nucleotides, Kozak sequence, and signal sequence. The final
multiepitope minigene is assembled by extending the overlapping
oligonucleotides in three sets of reactions using PCR. A
Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are
performed using the following conditions: 95.degree. C. for 15 sec,
annealing temperature (5.degree. below the lowest calculated Tm of
each primer pair) for 30 sec, and 72.degree. C. for 1 min.
[0601] For example, a minigene is prepared as follows. For a first
PCR reaction, 5 .mu.g of each of two oligonucleotides are annealed
and extended: In an example using eight oligonucleotides, i.e.,
four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are
combined in 100 .mu.l reactions containing Pfu polymerase buffer
(1.times.=10 mM KCL, 10 mM (NH4).sub.2SO.sub.4, 20 mM
Tris-chloride, pH 8.75, 2 mM MgSO.sub.4 0.1% Triton X-100, 100
.mu.g/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The
full-length dimer products are gel-purified, and two reactions
containing the product of 1+2 and 3+4, and the product of 5+6 and
7+8 are mixed, annealed, and extended for 10 cycles. Half of the
two reactions are then mixed, and 5 cycles of annealing and
extension carried out before flanking primers are added to amplify
the full length product. The full-length product is gel-purified
and cloned into pCR-blunt (Invitrogen) and individual clones are
screened by sequencing.
Example 21
[0602] The Plasmid Construct and the Degree to Which it Induces
Immunogenicity
[0603] The degree to which a plasmid construct, for example a
plasmid constructed in accordance with the previous Example, is
able to induce immunogenicity is confirmed in vitro by determining
epitope presentation by APC following transduction or transfection
of the APC with an epitope-expressing nucleic acid construct. Such
a study determines "antigenicity" and allows the use of human APC.
The assay determines the ability of the epitope to be presented by
the APC in a context that is recognized by a T cell by quantifying
the density of epitope-HLA class I complexes on the cell surface.
Quantitation can be performed by directly measuring the amount of
peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol.
156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the
number of peptide-HLA class I complexes can be estimated by
measuring the amount of lysis or lymphokine release induced by
diseased or transfected target cells, and then determining the
concentration of peptide necessary to obtain equivalent levels of
lysis or lymphokine release (see, e.g., Kageyama et al., J.
Immunol. 154:567-576, 1995).
[0604] Alternatively, immunogenicity is confirmed through in vivo
injections into mice and subsequent in vitro assessment of CTL and
HTL activity, which are analyzed using cytotoxicity and
proliferation assays, respectively, as detailed e.g., in Alexander
et al., Immunity 1:751-761, 1994.
[0605] For example, to confirm the capacity of a DNA minigene
construct containing at least one HLA-A2 supermotif peptide to
induce CTLs in vivo, HLA-A2.1/K.sup.b transgenic mice, for example,
are immunized intramuscularly with 100 .mu.g of naked cDNA. As a
means of comparing the level of CTLs induced by cDNA immunization,
a control group of animals is also immunized with an actual peptide
composition that comprises multiple epitopes synthesized as a
single polypeptide as they would be encoded by the minigene.
[0606] Splenocytes from immunized animals are stimulated twice with
each of the respective compositions (peptide epitopes encoded in
the minigene or the polyepitopic peptide), then assayed for
peptide-specific cytotoxic activity in a .sup.51Cr release assay.
The results indicate the magnitude of the CTL response directed
against the A2-restricted epitope, thus indicating the in vivo
immunogenicity of the minigene vaccine and polyepitopic
vaccine.
[0607] It is, therefore, found that the minigene elicits immune
responses directed toward the HLA-A2 supermotif peptide epitopes as
does the polyepitopic peptide vaccine. A similar analysis is also
performed using other HLA-A3 and HLA-B7 transgenic mouse models to
assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif
epitopes, whereby it is also found that the minigene elicits
appropriate immune responses directed toward the provided
epitopes.
[0608] To confirm the capacity of a class II epitope-encoding
minigene to induce HTLs in vivo, DR transgenic mice, or for those
epitopes that cross react with the appropriate mouse MHC molecule,
I-A.sup.b-restricted mice, for example, are immunized
intramuscularly with 100 .mu.g of plasmid DNA. As a means of
comparing the level of HTLs induced by DNA immunization, a group of
control animals is also immunized with an actual peptide
composition emulsified in complete Freund's adjuvant. CD4+ T cells,
ie. HTLs, are purified from splenocytes of immunized animals and
stimulated with each of the respective compositions (peptides
encoded in the minigene). The HTL response is measured using a
.sup.3H-thymidine incorporation proliferation assay, (see, e.g.,
Alexander et al. Immunity 1:751-761, 1994). The results indicate
the magnitude of the HTL response, thus demonstrating the in vivo
immunogenicity of the minigene.
[0609] DNA minigenes, constructed as described in the previous
Example, can also be confirmed as a vaccine in combination with a
boosting agent using a prime boost protocol. The boosting agent can
consist of recombinant protein (e.g., Barnett et al., Aids Res. and
Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant
vaccinia, for example, expressing a minigene or DNA encoding the
complete protein of interest (see, e.g., Hanke et al., Vaccine
16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci USA
95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181,
1999; and Robinson et al., Nature Med. 5:526-34, 1999).
[0610] For example, the efficacy of the DNA minigene used in a
prime boost protocol is initially evaluated in transgenic mice. In
this example, A.sub.2.1/K.sup.b transgenic mice are immunized IM
with 100 .mu.g of a DNA minigene encoding the immunogenic peptides
including at least one HLA-A2 supermotif-bearing peptide. After an
incubation period (ranging from 3-9 weeks), the mice are boosted IP
with 10.sup.7 pfu/mouse of a recombinant vaccinia virus expressing
the same sequence encoded by the DNA minigene. Control mice are
immunized with 100 .mu.g of DNA or recombinant vaccinia without the
minigene sequence, or with DNA encoding the minigene, but without
the vaccinia boost. After an additional incubation period of two
weeks, splenocytes from the mice are immediately assayed for
peptide-specific activity in an ELISPOT assay. Additionally,
splenocytes are stimulated in vitro with the A2-restricted peptide
epitopes encoded in the minigene and recombinant vaccinia, then
assayed for peptide-specific activity in an alpha, beta and/or
gamma IFN ELISA.
[0611] It is found that the minigene utilized in a prime-boost
protocol elicits greater immune responses toward the HLA-A2
supermotif peptides than with DNA alone. Such an analysis can also
be performed using HLA-A11 or HLA-B7 transgenic mouse models to
assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif
epitopes. The use of prime boost protocols in humans is described
below in the Example entitled "Induction of CTL Responses Using a
Prime Boost Protocol."
Example 22
[0612] Peptide Composition for Prophylactic Uses
[0613] Vaccine compositions of the present invention can be used to
prevent 158P1H4 expression in persons who are at risk for tumors
that bear this antigen. For example, a polyepitopic peptide epitope
composition (or a nucleic acid comprising the same) containing
multiple CTL and HTL epitopes such as those selected in the above
Examples, which are also selected to target greater than 80% of the
population is administered to individuals at risk for a
158P1H4-associated tumor.
[0614] For example, a peptide-based composition is provided as a
single polypeptide that encompasses multiple epitopes. The vaccine
is typically administered in a physiological solution that
comprises an adjuvant, such as Incomplete Freunds Adjuvant. The
dose of peptide for the initial immunization is from about 1 to
about 50,000 .mu.g, generally 100-5,000 .mu.g, for a 70 kg patient.
The initial administration of vaccine is followed by booster
dosages at 4 weeks followed by evaluation of the magnitude of the
immune response in the patient, by techniques that determine the
presence of epitope-specific CTL populations in a PBMC sample.
Additional booster doses are administered as required. The
composition is found to be both safe and efficacious as a
prophylaxis against 158P1H4-associated disease.
[0615] Alternatively, a composition typically comprising
transfecting agents is used for the administration of a nucleic
acid-based vaccine in accordance with methodologies known in the
art and disclosed herein.
Example 23
[0616] Polyepitopic Vaccine Compositions Derived from Native
158P1H4 Sequences
[0617] A native 158P1H4 polyprotein sequence is analyzed,
preferably using computer algorithms defined for each class I
and/or class II supermotif or motif, to identify "relatively short"
regions of the polyprotein that comprise multiple epitopes. The
"relatively short" regions are preferably less in length than an
entire native antigen. This relatively short sequence that contains
multiple distinct or overlapping, "nested" epitopes is selected; it
can be used to generate a minigene construct. The construct is
engineered to express the peptide, which corresponds to the native
protein sequence. The "relatively short" peptide is generally less
than 250 amino acids in length, often less than 100 amino acids in
length, preferably less than 75 amino acids in length, and more
preferably less than 50 amino acids in length. The protein sequence
of the vaccine composition is selected because it has maximal
number of epitopes contained within the sequence, i.e., it has a
high concentration of epitopes. As noted herein, epitope motifs may
be nested or overlapping (i.e., frame shifted relative to one
another). For example, with overlapping epitopes, two 9-mer
epitopes and one 10-mer epitope can be present in a 10 amino acid
peptide. Such a vaccine composition is administered for therapeutic
or prophylactic purposes.
[0618] The vaccine composition will include, for example, multiple
CTL epitopes from 158P1H4 antigen and at least one HTL epitope.
This polyepitopic native sequence is administered either as a
peptide or as a nucleic acid sequence which encodes the peptide.
Alternatively, an analog can be made of this native sequence,
whereby one or more of the epitopes comprise substitutions that
alter the cross-reactivity and/or binding affinity properties of
the polyepitopic peptide.
[0619] The embodiment of this example provides for the possibility
that an as yet undiscovered aspect of immune system processing will
apply to the native nested sequence and thereby facilitate the
production of therapeutic or prophylactic immune response-inducing
vaccine compositions. Additionally such an embodiment provides for
the possibility of motif-bearing epitopes for an HLA makeup that is
presently unknown. Furthermore, this embodiment (excluding an
analoged embodiment) directs the immune response to multiple
peptide sequences that are actually present in native 158P1H4, thus
avoiding the need to evaluate any junctional epitopes. Lastly, the
embodiment provides an economy of scale when producing peptide or
nucleic acid vaccine compositions.
[0620] Related to this embodiment, computer programs are available
in the art which can be used to identify in a target sequence, the
greatest number of epitopes per sequence length.
Example 24
[0621] Polyepitopic Vaccine Compositions from Multiple Antigens
[0622] The 158P1 H4 peptide epitopes of the present invention are
used in conjunction with epitopes from other target
tumor-associated antigens, to create a vaccine composition that is
useful for the prevention or treatment of cancer that expresses
158P1H4 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 158P1H4 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 158P1H4
expression, or can be administered as a composition comprising a
cocktail of one or more discrete epitopes. Alternatively, the
vaccine can be administered as a minigene construct or as dendritic
cells which have been loaded with the peptide epitopes in
vitro.
Example 25
[0623] Use of Peptides to Evaluate an Immune Response
[0624] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 158P1H4. Such an analysis can be performed in a manner
described by Ogg et al., Science 279:2103-2106, 1998. In this
Example, peptides in accordance with the invention are used as a
reagent for diagnostic or prognostic purposes, not as an
immunogen.
[0625] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 158P1H4 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising an 158P1H4
peptide containing an A*0201 motif. Tetrameric complexes are
synthesized as described (Musey et al., N. Engl. J. Med. 337:1267,
1997). Briefly, purified HLA heavy chain (A*0201 in this example)
and .beta.2-microglobulin are synthesized by means of a prokaryotic
expression system. The heavy chain is modified by deletion of the
transmembrane-cytosolic tail and COOH-terminal addition of a
sequence containing a BirA enzymatic biotinylation site. The heavy
chain, .beta.2-microglobulin, and peptide are refolded by dilution.
The 45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerytbrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0626] For the analysis of patient blood samples, approximately one
million PBMCs are centrifuged at 300 g for 5 minutes and
resuspended in 50 .mu.l of cold phosphate-buffered saline.
Tri-color analysis is performed with the tetramer-phycoerythrin,
along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are
incubated with tetramer and antibodies on ice for 30 to 60 min and
then washed twice before formaldehyde fixation. Gates are applied
to contain >99.98% of control samples. Controls for the
tetramers include both A*0201-negative individuals and
A*0201-positive non-diseased donors. The percentage of cells
stained with the tetramer is then determined by flow cytometry. The
results indicate the number of cells in the PBMC sample that
contain epitope-restricted CTLs, thereby readily indicating the
extent of immune response to the 158P1H4 epitope, and thus the
status of exposure to 158P1H4, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 26
[0627] Use of Peptide Epitopes to Evaluate Recall Responses
[0628] The peptide epitopes of the invention are used as reagents
to evaluate T cell responses, such as acute or recall responses, in
patients. Such an analysis may be performed on patients who have
recovered from 158P1H4-associated disease or who have been
vaccinated with an 158P1H4 vaccine.
[0629] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
158P1H4 vaccine. PBMC are collected from vaccinated individuals and
HLA typed. Appropriate peptide epitopes of the invention that,
optimally, bear supermotifs to provide cross-reactivity with
multiple HLA supertype family members, are then used for analysis
of samples derived from individuals who bear that HLA type.
[0630] PBMC from vaccinated individuals are separated on
Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis,
Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended
in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2
mM), penicillin (50 U/ml), streptomycin (50 .mu.g/ml), and Hepes
(10 mM) containing 10% heat-inactivated human AB serum (complete
RPMI) and plated using microculture formats. A synthetic peptide
comprising an epitope of the invention is added at 10 .mu.g/ml to
each well and HBV core 128-140 epitope is added at 1 .mu.g/ml to
each well as a source of T cell help during the first week of
stimulation.
[0631] In the microculture format, 4.times.10.sup.5 PBMC are
stimulated with peptide in 8 replicate cultures in 96-well round
bottom plate in 100 .mu.l/ well of complete RPMI. On days 3 and 10,
100 ul of complete RPMI and 20 U/ml final concentration of rIL-2
are added to each well. On day 7 the cultures are transferred into
a 96-well flat-bottom plate and restimulated with peptide, rIL-2
and 10.sup.5 irradiated (3,000 rad) autologous feeder cells. The
cultures are tested for cytotoxic activity on day 14. A positive
CTL response requires two or more of the eight replicate cultures
to display greater than 10% specific .sup.51Cr release, based on
comparison with non-diseased control subjects as previously
described (Rehermann, et al., Nature Med. 2:1104,1108, 1996;
Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and
Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).
[0632] Target cell lines are autologous and allogeneic
EBV-transformed B-LCL that are either purchased from the American
Society for Histocompatibility and Immunogenetics (ASHI, Boston,
Mass.) or established from the pool of patients as described
(Guilhot, et al. J. Virol. 66:2670-2678, 1992).
[0633] Cytotoxicity assays are performed in the following manner.
Target cells consist of either allogeneic HLA-matched or autologous
EBV-transformed B lymphoblastoid cell line that are incubated
overnight with the synthetic peptide epitope of the invention at 10
.mu.M, and labeled with 100 .mu.Ci of .sup.51Cr (Amersham Corp.,
Arlington Heights, Ill.) for 1 hour after which they are washed
four times with HBSS.
[0634] Cytolytic activity is determined in a standard 4-h, split
well .sup.51Cr release assay using U-bottomed 96 well plates
containing 3,000 targets/well. Stimulated PBMC are tested at
effector/target (E/T) ratios of 20-50:1 on day 14. Percent
cytotoxicity is determined from the formula:
100.times.[(experimental release-spontaneous release)/maximum
release-spontaneous release)]. Maximum release is determined by
lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co.,
St. Louis, Mo.). Spontaneous release is <25% of maximum release
for all experiments.
[0635] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 158P1H4 or an 158P1H4 vaccine.
[0636] Similarly, Class II restricted HTL responses may also be
analyzed. Purified PBMC are cultured in a 96-well flat bottom plate
at a density of 1.5.times.10.sup.5 cells/well and are stimulated
with 10 .mu.g/ml synthetic peptide of the invention, whole 158P1H4
antigen, or PHA. Cells are routinely plated in replicates of 4-6
wells for each condition. After seven days of culture, the medium
is removed and replaced with fresh medium containing 10 U/ml IL-2.
Two days later, 1 .mu.Ci .sup.3H-thymidine is added to each well
and incubation is continued for an additional 18 hours. Cellular
DNA is then harvested on glass fiber mats and analyzed for
.sup.3H-thymidine incorporation. Antigen-specific T cell
proliferation is calculated as the ratio of .sup.3H-thymidine
incorporation in the presence of antigen divided by the
.sup.3H-thymidine incorporation in the absence of antigen.
Example 27
[0637] Induction of Specific CTL Response in Humans
[0638] A human clinical trial for an immunogenic composition
comprising CTL and HTL epitopes of the invention is set up as an
IND Phase I, dose escalation study and carried out as a randomized,
double-blind, placebo-controlled trial. Such a trial is designed,
for example, as follows:
[0639] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0640] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0641] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0642] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0643] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0644] The endpoints measured in this study relate to the safety
and tolerability of the peptide composition as well as its
immunogenicity. Cellular immune responses to the peptide
composition are an index of the intrinsic activity of this the
peptide composition, and can therefore be viewed as a measure of
biological efficacy. The following summarize the clinical and
laboratory data that relate to safety and efficacy endpoints.
[0645] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0646] Evaluation of Vaccine Efficacy: For evaluation of vaccine
efficacy, subjects are bled before and after injection. Peripheral
blood mononuclear cells are isolated from fresh heparinized blood
by Ficoll-Hypaque density gradient centrifugation, aliquoted in
freezing media and stored frozen. Samples are assayed for CTL and
HTL activity.
[0647] The vaccine is found to be both safe and efficacious.
Example 28
[0648] Phase II Trials in Patients Expressing 158P1H4
[0649] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 158P1H4. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 158P1H4, to establish the safety of
inducing a CTL and HTL response in these patients, and to see to
what extent activation of CTLs improves the clinical picture of
these patients, as manifested, e.g., by the reduction and/or
shrinking of lesions. Such a study is designed, for example, as
follows:
[0650] The studies are performed in multiple centers. The trial
design is an open-label, uncontrolled, dose escalation protocol
wherein the peptide composition is administered as a single dose
followed six weeks later by a single booster shot of the same dose.
The dosages are 50, 500 and 5,000 micrograms per injection.
Drug-associated adverse effects (severity and reversibility) are
recorded.
[0651] There are three patient groupings. The first group is
injected with 50 micrograms of the peptide composition and the
second and third groups with 500 and 5,000 micrograms of peptide
composition, respectively. The patients within each group range in
age from 21-65 and represent diverse ethnic backgrounds. All of
them have a tumor that expresses 158P1H4.
[0652] Clinical manifestations or antigen-specific T-cell responses
are monitored to assess the effects of administering the peptide
compositions. The vaccine composition is found to be both safe and
efficacious in the treatment of 158P1H4-associated disease.
Example 29
[0653] Induction of CTL Responses Using a Prime Boost Protocol
[0654] A prime boost protocol similar in its underlying principle
to that used to confirm the efficacy of a DNA vaccine in transgenic
mice, such as described above in the Example entitled "The Plasmid
Construct and the Degree to Which It Induces Immunogenicity," can
also be used for the administration of the vaccine to humans. Such
a vaccine regimen can include an initial administration of, for
example, naked DNA followed by a boost using recombinant virus
encoding the vaccine, or recombinant protein/polypeptide or a
peptide mixture administered in an adjuvant.
[0655] For example, the initial immunization may be performed using
an expression vector, such as that constructed in the Example
entitled "Construction of `Minigene` Multi-Epitope DNA Plasmids" in
the form of naked nucleic acid administered IM (or SC or ID) in the
amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to
1000 .mu.g) can also be administered using a gene gun. Following an
incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-107 to 5.times.10.sup.9 pfu. An
alternative recombinant virus, such as an MVA, canarypox,
adenovirus, or adeno-associated virus, can also be used for the
booster, or the polyepitopic protein or a mixture of the peptides
can be administered. For evaluation of vaccine efficacy, patient
blood samples are obtained before immunization as well as at
intervals following administration of the initial vaccine and
booster doses of the vaccine. Peripheral blood mononuclear cells
are isolated from fresh heparinized blood by Ficoll-Hypaque density
gradient centrifugation, aliquoted in freezing media and stored
frozen. Samples are assayed for CTL and HTL activity.
[0656] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 158P1H4 is generated.
Example 30
[0657] Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0658] Vaccines comprising peptide epitopes of the invention can be
administered using APCs, or "professional" APCs such as DC. In this
example, peptide-pulsed DC are administered to a patient to
stimulate a CTL response in vivo. In this method, dendritic cells
are isolated, expanded, and pulsed with a vaccine comprising
peptide CTL and HTL epitopes of the invention. The dendritic cells
are infused back into the patient to elicit CTL and HTL responses
in vivo. The induced CTL and HTL then destroy or facilitate
destruction, respectively, of the target cells that bear the
158P1H4 protein from which the epitopes in the vaccine are
derived.
[0659] For example, a cocktail of epitope-comprising peptides is
administered ex vivo to PBMC, or isolated DC therefrom. A
pharmaceutical to facilitate harvesting of DC can be used, such as
Progenipoietin.TM. (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After
pulsing the DC with peptides, and prior to reinfusion into
patients, the DC are washed to remove unbound peptides.
[0660] As appreciated clinically, and readily determined by one of
skill based on clinical outcomes, the number of DC reinfused into
the patient can vary (see, e.g., Nature Med. 4:328, 1998; Nature
Med. 2:52, 1996 and Prostate 32:272, 1997). Although
2.times.50.times.10.sup.6 DC per patient are typically
administered, larger number of DC, such as 10.sup.7 or 10.sup.8 can
also be provided. Such cell populations typically contain between
50-90% DC.
[0661] In some embodiments, peptide-loaded PBMC are injected into
patients without purification of the DC. For example, PBMC
generated after treatment with an agent such as Progenipoietin.TM.
are injected into patients without purification of the DC. The
total number of PBMC that are administered often ranges from
10.sup.8 to 10.sup.10. Generally, the cell doses injected into
patients is based on the percentage of DC in the blood of each
patient, as determined, for example, by immunofluorescence analysis
with specific anti-DC antibodies. Thus, for example, if
Progenipoietin.TM. mobilizes 2% DC in the peripheral blood of a
given patient, and that patient is to receive 5.times.10.sup.6 DC,
then the patient will be injected with a total of
2.5.times.10.sup.8 peptide-loaded PBMC. The percent DC mobilized by
an agent such as Progenipoietin.TM. is typically estimated to be
between 2-10%, but can vary as appreciated by one of skill in the
art.
[0662] Ex vivo Activation of CTL/HTL Responses
[0663] Alternatively, ex vivo CTL or HTL responses to 158P1H4
antigens can be induced by incubating, in tissue culture, the
patient's, or genetically compatible, CTL or HTL precursor cells
together with a source of APC, such as DC, and immunogenic
peptides. After an appropriate incubation time (typically about
7-28 days), in which the precursor cells are activated and expanded
into effector cells, the cells are infused into the patient, where
they will destroy (CTL) or facilitate destruction (HTL) of their
specific target cells, i.e., tumor cells.
Example 31
[0664] An Alternative Method of Identifying and Confirming
Motif-Bearing Peptides
[0665] Another method of identifying and confirming motif-bearing
peptides is to elute them from cells bearing defined MHC molecules.
For example, EBV transformed B cell lines used for tissue typing
have been extensively characterized to determine which HLA
molecules they express. In certain cases these cells express only a
single type of HLA molecule. These cells can be transfected with
nucleic acids that express the antigen of interest, e.g. 158P1H4.
Peptides produced by endogenous antigen processing of peptides
produced as a result of transfection will then bind to HLA
molecules within the cell and be transported and displayed on the
cell's surface. Peptides are then eluted from the HLA molecules by
exposure to mild acid conditions and their amino acid sequence
determined, e.g., by mass spectral analysis (e.g., Kubo et al., J.
Immunol. 152:3913, 1994). Because the majority of peptides that
bind a particular HLA molecule are motif-bearing, this is an
alternative modality for obtaining the motif-bearing peptides
correlated with the particular HLA molecule expressed on the
cell.
[0666] Alternatively, cell lines that do not express endogenous HLA
molecules can be transfected with an expression construct encoding
a single HLA allele. These cells can then be used as described,
i.e., they can then be transfected with nucleic acids that encode
158P1H4 to isolate peptides corresponding to 158P1H4 that have been
presented on the cell surface. Peptides obtained from such an
analysis will bear motif(s) that correspond to binding to the
single HLA allele that is expressed in the cell.
[0667] As appreciated by one in the art, one can perform a similar
analysis on a cell bearing more than one HLA allele and
subsequently determine peptides specific for each HLA allele
expressed. Moreover, one of skill would also recognize that means
other than transfection, such as loading with a protein antigen,
can be used to provide a source of antigen to the cell.
Example 32
[0668] Complementary Polynucleotides
[0669] Sequences complementary to the 158P1H4-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 158P1H4. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using, e.g., OLIGO 4.06 software (National Biosciences)
and the coding sequence of 158P1H4. To inhibit transcription, a
complementary oligonucleotide is designed from the most unique 5'
sequence and used to prevent promoter binding to the coding
sequence. To inhibit translation, a complementary oligonucleotide
is designed to prevent ribosomal binding to the 158P1H4-encoding
transcript.
Example 33
[0670] Purification of Naturally-occurring or Recombinant 158P1H4
Using 158P1H4 Specific Antibodies
[0671] Naturally occurring or recombinant 158P1H4 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 158P1H4. An immunoaffinity column is constructed by covalently
coupling anti-158P1H4 antibody to an activated chromatographic
resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia
Biotech). After the coupling, the resin is blocked and washed
according to the manufacturer's instructions.
[0672] Media containing 158P1H4 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 158P1H4 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/158P1H4 binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and GCR.P is collected.
Example 34
[0673] Identification of Molecules Which Interact with 158P1H4
[0674] 158P1H4, or biologically active fragments thereof, are
labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
158P1H4, washed, and any wells with labeled 158P1H4 complex are
assayed. Data obtained using different concentrations of 158P1H4
are used to calculate values for the number, affinity, and
association of 158P1H4 with the candidate molecules. Throughout
this application, various website data content, publications,
applications and patents are referenced. (Websites are referenced
by their Uniform Resource Locator, or URL, addresses on the World
Wide Web.) The disclosures of each of these items of information
are are hereby incorporated by reference herein in their
entireties.
Example 35
[0675] In Vivo Assay for 158P1H4 Tumor Growth Promotion
[0676] The effect of the 158P1H4 protein on tumor cell growth can
be confirmed in vivo by gene overexpression in bladder cancer
cells. For example, SCID mice can be injected SQ on each flank with
1.times.10.sup.6 bladder cancer cells (such as SCaBER, UM-UC-3,
HT1376, RT4, T24, TCC-SUP, J82 and SW780 cells) containing tkNeo
empty vector or 158P1H4.
[0677] At least two strategies may be used: (1) Constitutive
158P1H4 expression under regulation of a promoter such as a
constitutive promoter obtained from the genomes of viruses such as
polyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), or from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, provided
such promoters are compatible with the host cell systems. (2)
Regulated expression under control of an inducible vector system,
such as ecdysone, tet, etc., can be used provided such promoters
are compatible with the host cell systems. Tumor volume is then
monitored at the appearance of palpable tumors and is followed over
time to determine if 158P1H4-expressing cells grow at a faster rate
and whether tumors produced by 158P1H4-expressing cells demonstrate
characteristics of altered aggressiveness (e.g. enhanced
metastasis, vascularization, reduced responsiveness to
chemotherapeutic drugs). Additionally, mice can be implanted with
the same cells orthotopically to determine if 158P1H4 has an effect
on local growth in the bladder or on the ability of the cells to
metastasize, specifically to lungs or lymph nodes (Fu, X., et al.,
Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer
Res., 1997. 17: p. 3239-3242; Peralta, E. A., et al., J. Urol.,
1999. 162: p. 1806-1811). Furthermore, this assay is useful to
confirm the 158P1H4 inhibitory effect of candidate therapeutic
compositions, such as for example, 158P1H4 antibodies or
intrabodies, and 158P1H4 antisense molecules or ribozymes.
Example 36
[0678] 158P1H4 Monoclonal Antibody-mediated Inhibition of Bladder
Tumors In Vivo
[0679] The significant expression of 158P1H4 in cancer tissues,
together with its restricted expression in normal tissues, makes
158P1H4 an excellent target for antibody therapy. In cases where
the monoclonal antibody target is a cell surface protein,
antibodies have been shown to be efficacious at inhibiting tumor
growth (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or
www.pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the
target is not on the cell surface, such as for 158P1H4 and
including PSA and PAP in prostate cancer, antibodies have still
been shown to recognize and inhibit growth of cells expressing
those proteins (Saffran, D. C., et al., Cancer and Metastasis
Reviews, 1999. 18: p. 437-449). As with any cellular protein with a
restricted expresson profile, 158P1H4 is a target for T cell-based
immunotherapy.
[0680] Accordingly, the therapeutic efficacy of anti-158P1H4 mAbs
in human bladder cancer mouse models is modeled in
158P1H4-expressing bladder cancer xenografts or bladder cancer cell
lines, such as those described in Example (the Example entitled "In
Vivo Assay for 158P1H4 Tumor Growth Promotion", that have been
engineered to express 158P1H4.
[0681] Antibody efficacy on tumor growth and metastasis formation
is confirmed, e.g., in a mouse orthotopic bladder cancer xenograft
model. The antibodies can be unconjugated, as discussed in this
Example, or can be conjugated to a therapeutic modality, as
appreciated in the art. It is confirmed that anti-158P1H4 mAbs
inhibit formation of 158P1H4-expressing bladder tumors.
Anti-158P1H4 mAbs also retard the growth of established orthotopic
tumors and prolong survival of tumor-bearing mice. These results
indicate the utility of anti-158P1H4 mAbs in the treatment of local
and advanced stages of bladder cancer. (See, e.g., Saffran, D., et
al., PNAS 10:1073-1078 or
www.pnas.org/cgi/doi/10.1073/pnas.051624698)
[0682] Administration of anti-158P1H4 mAbs retard established
orthotopic tumor growth and inhibit metastasis to distant sites,
resulting in a significant prolongation in the survival of
tumor-bearing mice. These studies indicate that 158P1H4 is an
attractive target for immunotherapy and demonstrate the therapeutic
potential of anti-158P1H4 mAbs for the treatment of local and
metastatic bladder cancer.
[0683] This example demonstrates that unconjugated 158P1H4
monoclonal antibodies effectivly to inhibit the growth of human
bladder tumors grown in SCID mice; accordingly a combination of
such efficacious monoclonal antibodies is also effective.
[0684] Tumor Inhibition Using Multiple Unconjugated 158P1H4 mAbs
Materials and Methods
[0685] 158P1H4 Monoclonal Antibodies
[0686] Monoclonal antibodies are raised against 158P1H4 as
described in the Example entitled "Generation of 158P1H4 Monoclonal
Antibodies (mAbs)." The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation, in accordance with
techniques known in the art, for their capacity to bind 158P1H4.
Epitope mapping data for the anti-158P1H4 mAbs, as determined by
ELISA and Western analysis, recognize epitopes on the 158P1H4
protein. Immunohistochemical analysis of bladder cancer tissues and
cells with these antibodies is performed.
[0687] The monoclonal antibodies are purified from ascites or
hybridoma tissue culture supernatants by Protein-G Sepharose
chromatography, dialyzed against PBS, filter sterilized, and stored
at -20.degree. C. Protein determinations are performed by a
Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic
monoclonal antibody or a cocktail comprising a mixture of
individual monoclonal antibodies is prepared and used for the
treatment of mice receiving subcutaneous or orthotopic injections
of bladder tumor xenografts.
[0688] Bladder Cancer Cell Lines
[0689] Bladder cancer cell lines (Scaber, J82, UM-UC-3, HT1376,
RT4, T24, TCC-SUP, J82 and SW780) expressing 158P1H4 are generated
by retroviral gene transfer as described in Hubert, R. S., et al.,
STEAP: a prostate-specific cell-surface antigen highly expressed in
human prostate tumors. Proc Natl Acad Sci U S A, 1999.
96(25):14523-8. Anti-158P1H4 staining is detected by using an
FITC-conjugated goat anti-mouse antibody (Southern Biotechnology
Associates) followed by analysis on a Coulter Epics-XL f low
cytometer.
[0690] In Vivo Mouse Models
[0691] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10.sup.6 158P1H4-expressing bladder cancer cells mixed at a
1:1 dilution with Matrigel (Collaborative Research) in the right
flank of male SCID mice. To test antibody efficacy on tumor
formation, i.p. antibody injections are started on the same day as
tumor-cell injections. As a control, mice are injected with either
purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody
that recognizes an irrelevant antigen not expressed in human cells.
In preliminary studies, no difference is found between mouse IgG or
PBS on tumor growth. Tumor sizes are determined by vernier caliper
measurements, and the tumor volume is calculated as
length.times.width.times.height. Mice with s.c. tumors greater than
1.5 cm in diameter are sacrificed. Circulating levels of
anti-158P1H4 mAbs are determined by a capture ELISA kit (Bethyl
Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al.,
PNAS 10:1073-1078)
[0692] Orthotopic injections are performed, for example, in two
alternative embodiments, under anesthesia by, for example, use of
ketamine/xylazine. In a first embodiment, an intravesicular
injection of bladder cancer cells is administered directly through
the urethra and into the bladder (Peralta, E. A., et al., J. Urol.,
1999. 162:1806-1811). In a second embodiment, an incision is made
through the abdominal wall, the bladder is exposed, and bladder
tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are
surgically glued onto the the exterior wall of the bladder, termed
"onplantation" (Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939;
Chang, S., et al., Anticancer Res., 1997. 17: p.3239-3242).
Antibodies can be administered to groups of mice at the time of
tumor injection or onplantation, or after 1-2 weeks to allow tumor
establishment.
[0693] Anti-158P1H4 mAbs Inhibit Growth of 158P1H4-Expressing
Bladder Cancer Tumors
[0694] In one embodiment, the effect of anti-158P1H4 mAbs on tumor
formation is tested by using the bladder onplantation orthotopic
model. As compared with the s.c. tumor model, the orthotopic model,
which requires surgical attachment of tumor tissue directly on the
bladder, results in a local tumor growth, development of metastasis
in distal sites, and subsequent death (Fu, X., et al., Int. J.
Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer Res.,
1997.17: p. 3239-3242). This features make the orthotopic model
more representative of human disease progression and allows one to
follow the therapeutic effect of mAbs, as well as other therapeutic
modalities, on clinically relevant end points.
[0695] Accordingly, 158P1H4-expressing tumor cells are onplanted
orthotopically, and 2 days later, the mice are segregated into two
groups and treated with either: a) 50-2000.mu.g, usually
200-500.mu.g, of anti-158P1H4 Ab, or b) PBS, three times per week
for two to five weeks. Mice are monitored weekly for indications of
tumor growth.
[0696] As noted, a major advantage of the orthotopic bladder cancer
model is the ability to study the development of metastases.
Formation of metastasis in mice bearing established orthotopic
tumors is studied by histological analysis of tissue sections,
including lung and lymph nodes (Fu, X., et al., Int. J. Cancer,
1991. 49:938-939; Chang, S., et al., Anticancer Res., 1997.
17:3239-3242). Additionally, IHC analysis using anti-158P1H4
antibodies can be performed on the tissue sections.
[0697] Mice bearing established orthotopic 158P1H4-expressing
bladder tumors are administered 1000 .mu.g injections of either
anti-158P1H4 mAb or PBS over a 4-week period. Mice in both groups
are allowed to establish a high tumor burden (1-2 weeks growth), to
ensure a high frequency of metastasis formation in mouse lungs and
lymph nodes. Mice are then sacrificed and their local bladder tumor
and lung and lymph node tissue are analyzed for the presence of
tumor cells by histology and IHC analysis.
[0698] These studies demonstrate a broad anti-tumor efficacy of
anti-158P1H4 antibodies on initiation and progression of bladder
cancer in mouse models. Anti-158P1H4 antibodies inhibit tumor
formation and retard the growth of already established tumors and
prolong the survival of treated mice. Moreover, anti-158P1H4 mAbs
demonstrate a dramatic inhibitory effect on the spread of local
bladder tumor to distal sites, even in the presence of a large
tumor burden. Thus, anti-158P1H4 mAbs are efficacious on major
clinically relevant end points including lessened tumor growth,
lessened metastasis, and prolongation of survival.
Example 37
[0699] Homology Comparison of 158P1H4 to Known Sequences
[0700] The 158P1H4 protein of FIG. 3 has 440 amino acids with
calculated molecular weight of 50.8 kDa, and pI of 6.6. 158P1H4 is
predicted to be a cytoplasmic protein, and to have two potential
cleavage sites at aa 50 and aa 238.
[0701] By use of the PubMed website of the N.C.B.I. available at
http://www.ncbi.nlm.nih.gov/entrez, it was found at the protein
level that 158P1H4 shows best homology to a putative mouse protein
AK014536 (PubMed record:
gi.vertline.12852456.vertline.dbj.vertline.BAB29419.1.ver- tline.),
with 75% identity and 86% homology. AK014536 is a putative mouse
protein of unknown function, that contains a PX (Phox) domain.
[0702] Additionally, it was found that the 158P1H4 protein shows
distinct homology to human sorting nexin 17 (PubMed record:
gi.vertline.14732569.vertline.ref.vertline.XP.sub.--033201.1.vertline.),
with 43% identity and 63% homology (see FIG. A), and some homology
to the human sorting nexin 2 protein (PubMed record:
gi.vertline.14725614.vertli-
ne.ref.vertline.XP.sub.--051176.1.vertline.), with 28% identity and
45% homology.
[0703] Sorting nexins (SNX) are hydrophilic proteins that associate
with the cell membrane, and play an important role in protein
trafficking and protein transport (Haft CR, et al. Mol Biol Cell.
2000, 12:4105-16; Horazdovsky BF et al. Mol Biol Cell. 1997,
8:1529-41). Most known sorting nexins have been found to associate
with cell surface receptors or other nexins in mammalian cells as
well as S. cerevisia. The hetero-multimeric complexes are
themselves targeted to the cell membrane via surface receptors
(Haft CR, Mol Biol Cell. 2000, 12:4105-16). Of particular relevance
to 158P1H4, SNX2 associates with the EGF receptor directing it to
lysosomes for degradation, thereby controlling receptor recycling
(Haft CR et al. Mol Cell Biol. 1998, 12:7278-87). SNX 1 regulates
membrane turnover by sequestering proteins into endosomal
compartments (Kurten RC et al, J Cell Sci. 2001, 114:1743).
[0704] All sorting nexins contain a relatively conserved region of
around 100 aa known as the Phox homology or PX domain (Ponting CP,
Protein Sci. 1996, 11:2353-7). Most of the homology between 158P1H
and SNX2 is limited to the PX domain as shown in FIG. B, with 28%
identity along that region. Although the function of the PX domain
is not completely understood, recent studies suggest that the PX
domain is necessary for the association of SNXs with the cell
membrane, and their interaction with cell surface receptors, such
as EGFR, PDGFR, insulin receptor and transferring receptor
(Phillips SA, et al. J Biol Chem. 2000, 276:5074). In addition, PX
domains are associated with intracellular signaling pathways,
including phospholipase D and PI3K (Ponting C.P. Protein Sci
1996;5:2353).
[0705] Our findings that 158P1H4 is overexpressed in bladder cancer
while showing a restricted expression pattern in normal tissues
suggests that the 158P1H4 gene may play an important role in
various cancers, including cancers of the bladder.
[0706] The tumor-associated expression profile of sorting nexins
has not been well studied in patient samples. However Northern blot
analysis of SNX5 shows alternatively spliced variants and
differential expression in human cancer cell lines (Otsuki T et al,
Biochem Biophys Res Comm. 1999, 265:630-635), suggesting that SNX
may contribute to the generation or progression of several human
cancers. Moreover, overexpression of SNX in engineered cells
results in reduced expression of EGFR, PGDFR and insulin receptor
(Kurten RC et al. Science 1996, 272:1008). This indicates that SNXs
regulate the expression, and therefore the function, of several
receptors that play an important role in tumor development and
progression.
[0707] It is provided by the present invention that 158P1H4
controls tumor growth and progression by regulating gene expression
as well as cell surface availability and recycling of key surface
proteins involved in tumor growth. In addition to controlling the
function of well-studied receptors such as EGFR and PDGFR, 158P1H4
is appears to regulate other growth factor receptors and receptors
involved in normal function of epithelial cells. Consistent with
this function, SNX1 was found to associate with the transferring
receptor (Haft CR et al. Mol Cell Biol. 1998, 12:7278-87), a
regulator of iron metabolism in mammalian cells. Accordingly, when
158P1H4 functions as a regulator of cell growth and apoptosis, or
protein expression and recycling, 158P1H4 is used for therapeutic,
diagnostic, prognostic or preventative purposes.
[0708] Example 38
[0709] Identification and Confirmation of Signal Transduction
Pathways
[0710] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J Neurochem. 2001; 76:217-223). In particular, sorting
nexin 3 has been reported to associate with protein kinases and
membrane proteins (Xu et al. Nat Cell Biol. 2001; 3:658). Using
immunoprecipitation and Western blotting techniques, proteins are
identified that associate with 158P1H4 and mediate signaling
events. Several pathways known to play a role in cancer biology can
be regulated by 158P1H4, including phospholipid pathways such as
PI3K, AKT, etc, adhesion and migration pathways, including FAK,
Rho, Rac-1, etc, as well as mitogenic/survival cascades such as
ERK, p38, etc (Cell Growth Differ. 2000,11:279; J Biol Chem. 1999,
274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).
Bioinformatic analysis revealed that 158P1H4 can become
phosphorylated by serine/threonine as well as tyrosine kinases.
Thus, the phosphorylation of 158P1H4 is provided by the present
invention to lead to activation of the above listed pathways.
[0711] Using, e.g., Western blotting techniques the ability of
158P1H4 to regulate these pathways is confirmed. Cells expressing
or lacking 158P1H4 are either left untreated or stimulated with
cytokines, androgen and anti-integrin antibodies. Cell lysates are
analyzed using anti-phospho-specific antibodies (Cell Signaling,
Santa Cruz Biotechnology) in order to detect phosphorylation and
regulation of ERK, p38, AKT, PI3K, PLC and other signaling
molecules. When 158P1H4 plays a role in the regulation of signaling
pathways, whether individually or communally, it is used as a
target for diagnostic, prognostic, preventative and therapeutic
purposes.
[0712] To confirm that 158P1H4 directly or indirectly activates
known signal transduction pathways in cells, luciferase (luc) based
transcriptional reporter assays are carried out in cells expressing
individual genes. These transcriptional reporters contain
consensus-binding sites for known transcription factors that lie
downstream of well-characterized signal transduction pathways. The
reporters and examples of these associated transcription factors,
signal transduction pathways, and activation stimuli are listed
below.
[0713] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0714] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0715] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0716] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0717] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0718] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0719] Gene-mediated effects can be assayed in cells showing mRNA
expression. Luciferase reporter plasmids can be introduced by
lipid-mediated transfection (TFX-50, Promega). Luciferase activity,
an indicator of relative transcriptional activity, is measured by
incubation of cell extracts with luciferin substrate and
luminescence of the reaction is monitored in a luminometer.
[0720] Signaling pathways activated by 158P1H4 are mapped and used
for the identification and validation of therapeutic targets. When
158P1H4 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and therapeutic purposes.
Example 39
[0721] Involvement in Tumor Progression
[0722] The 158P1H4 gene can contribute to the growth of cancer
cells. The role of 158P1H4 in tumor growth is confirmed in a
variety of primary and transfected cell lines including prostate,
colon, bladder and kidney cell lines as well as NIH 3T3 cells
engineered to stably express 158P1H4. Parental cells lacking
158P1H4 and cells expressing 158P1H4 are evaluated for cell growth
using a well-documented proliferation assay (Fraser S P, Grimes J
A, Djamgoz M B. Prostate. 2000;44:61, Johnson D E, Ochieng J, Evans
S L. Anticancer Drugs. 1996, 7:288).
[0723] To confirm the role of 158P1H4 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH3T3 cells lacking 158P1H4 are compared to NHI-3T3 cells
expressing 158P1H4, using a soft agar assay under stringent and
more permissive conditions (Song Z. et al. Cancer Res. 2000,
60:6730).
[0724] To confirm the role of 158P1H4 in invasion and metastasis of
cancer cells, a well-established assay is used, e.g., a Transwell
Insert System assay (Becton Dickinson) (Cancer Res. 1999, 59:6010).
Control cells, including prostate, colon, bladder and kidney cell
lines lacking 158P1H4 are compared to cells expressing 158P1H4.
Cells are loaded with the fluorescent dye, calcein, and plated in
the top well of the Transwell insert coated with a basement
membrane analog. Invasion is determined by fluorescence of cells in
the lower chamber relative to the fluorescence of the entire cell
population.
[0725] 158P1H4 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 158P1H4 are compared for
differences in cell cycle regulation using a well-established BrdU
assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short,
cells are grown under both optimal (full serum) and limiting (low
serum) conditions are labeled with BrdU and stained with anti-BrdU
Ab and propidium iodide. Cells are analyzed for entry into the G1,
S, and G2M phases of the cell cycle. Alternatively, the effect of
stress on apoptosis is evaluated in control parental cells and
cells expressing 158P1H4, including normal and tumor bladder cells.
Engineered and parental cells are treated with various
chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and
protein synthesis inhibitors, such as cycloheximide. Cells are
stained with annexin V-FITC and cell death is measured by FACS
analysis. The modulation of cell death by 158P1H4 can play a
critical role in regulating tumor progression and tumor load.
[0726] When 158P1H4 plays a role in cell growth, transformation,
invasion or apoptosis, it is used as a target for diagnostic,
prognostic, preventative and therapeutic purposes.
Example 40
[0727] Involvement in Angiogenesis
[0728] Angiogenesis or new capillary blood vessel formation is
necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996,
86:353; Folkman J. Endocrinology. 1998 139:441). Several assays
have been developed to measure angiogenesis in vitro and in vivo,
such as the tissue culture assays, endothelial cell tube formation,
and endothelial cell proliferation. Using these assays as well as
in vitro neo-vascularization, the effect of 158P1H4 on angiogenesis
is confirmed. For example, endothelial cells engineered to express
158P1H4 are evaluated using tube formation and proliferation
assays. The effect of 158P1H4 isalso confirmed in animal models in
vivo. For example, cells either expressing or lacking 158P1H4 are
implanted subcutaneously in immunocompromised mice. Endothelial
cell migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. When 158P1H4 affects angiogenesis,
it is used as a target for diagnostic, prognostic, preventative and
therapeutic purposes
Example 41
[0729] Regulation of Transcription
[0730] The localization of 158P1H4 in the cytosol, its similarity
to SNX which has been found to activate signalling pathways and to
regulate essential cellular functions, and the fact that it carries
a PX domain support the present invention use of 158P1H4 based on
its role in the transcriptional regulation of eukaryotic genes.
Regulation of gene expression is confirmed, e.g., by studying gene
expression in cells expressing or lacking 158P1H4. For this
purpose, two types of experiments are performed.
[0731] In the first set of experiments, RNA from parental and
158P1H4-expressing cells are extracted and hybridized to
commercially available gene arrays (Clontech) (Smid-Koopman E et
al. Br J Cancer. 2000. 83:246). Resting cells as well as cells
treated with FBS or androgen are compared. Differentially expressed
genes are identified in accordance with procedures known in the
art. The differentially expressed genes are then mapped to
biological pathways (Chen K et al., Thyroid. 2001. 11:41.).
[0732] In the second set of experiments, specific transcriptional
pathway activation is evaluated using commercially available
(Stratagene) luciferase reporter constructs including: NFkB-luc,
SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These
transcriptional reporters contain consensus binding sites for known
transcription factors that lie downstream of well-characterized
signal transduction pathways, and represent a good tool to
ascertain pathway activation and screen for positive and negative
modulators of pathway activation.
[0733] When 158P1H4 plays a role in gene regulation, it is used as
a target for diagnostic, prognostic, preventative and therapeutic
purposes.
Example 42
[0734] Involvement in Cell Adhesion
[0735] Cell adhesion plays a critical role in tissue colonization
and metastasis. Based on its homology to SNX, 158P1H4 can
participate in cellular organization, and as a consequence cell
adhesion and motility. To confirm that 158P1H4 regulates cell
adhesion, control cells lacking 158P1H4 are compared to cells
expressing 158P1H4, using techniques previously described (see,
e.g., Haier et al, Br. J. Cancer. 1999, 80:1867; Lehr and Pienta,
J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment,
cells labeled with a fluorescent indicator, such as calcein, are
incubated on tissue culture wells coated with media alone or with
matrix proteins. Adherent cells are detected by fluorimetric
analysis and percent adhesion is calculated. In another embodiment,
cells lacking or expressing 158P1H4 are analyzed for their ability
to mediate cell-cell adhesion using similar experimental techniques
as described above. Both of these experimental systems are used to
identify proteins, antibodies and/or small molecules that modulate
cell adhesion to extracellular matrix and cell-cell interaction.
Since cell adhesion plays a critical role in tumor growth,
progression, and, colonization, when 158P1H4 is involved in cell
adhesion, it serves as a diagnostic, prognostic, preventative and
therapeutic modality
Example 43
[0736] Involvement of 158P1H4 in Protein Trafficking
[0737] Due to its similarity to SNX, 158P1H4 can regulate
intracellular trafficking and retention into endosomal
compartments. Its role in the trafficking of proteins can be
confirmed using well-established methods (Valetti C. et al. Mol
Biol Cell. 1999, 10:4107). For example, FITC-conjugated
.alpha.2-macroglobulin is incubated with 158P1H4-expressing and
158P1H4-negative cells. The location and uptake of
FITC-.alpha.2-macroglobulin is visualized using a fluorescent
microscope. In another approach, the co-localization of 158P1H4
with vesicular proteins is confirmed by co-precipitation and
Western blotting techniques and fluorescent microscopy.
[0738] Alternatively, 158P1H4-expressing and 158P1H4-lacking cells
are compared using bodipy-ceramide labeled bovine serum albumine
(Huber L et al. Mol. Cell. Biol. 1995, 15:918). Briefly, cells are
allowed to take up the labeled BSA and are placed intermittently at
4.degree. C. and 18.degree. C. to allow for trafficking to take
place. Cells are examined under fluorescent microscopy, at
different time points, for the presence of labeled BSA in specific
vesicular compartments, including Golgi, endoplasmic reticulum,
etc.
[0739] In another embodiment, the effect of 158P1H4 on membrane
transport is examined using biotin-avidin complexes. Cells either
expressing or lacking 158P1H4 are transiently incubated with
biotin. The cells are placed at 4.degree. C. or transiently warmed
to 37.degree. C. for various periods of time. The cells are
fractionated and examined by avidin affinity precipitation for the
presence of biotin in specific cellular compartments. Using such
assay systems, proteins, antibodies and small molecules are
identified that modify the effect of 158P1H4 on vesicular
transport. When 158P1H4 plays a role in intracellular trafficking,
158P1H4 is a target for diagnostic, prognostic, preventative and
therapeutic purposes
Example 44
[0740] Protein-Protein Association
[0741] The two SNX proteins with homology to 158P1H4 have been
shown to interact with other proteins, thereby forming protein
complexes that can regulate protein localization, gene
transcription, and potentially cell transformation (Fucks, U. et
al, Proc Natl Acad Sci U S A. 2001, 98:8756; Haft C et al, Mol Cell
Biol. 1998, 18:7278). Using immunoprecipitation techniques as well
as two yeast hybrid systems, proteins are identified that associate
with 158P1H4. Immunoprecipitates from cells expressing 158P1H4 and
cells lacking 158P1H4 are compared for specific protein-protein
associations.
[0742] Studies are performed to determine the extent of the
association of 158P1H4 with receptors, such as the EGF and IGF
receptors. Studies comparing 158P1H4 positive and 158P1H4 negative
cells, as well as studies comparing unstimulated/resting cells and
cells treated with epithelial cell activators, such as cytokines,
growth factors and anti-integrin Ab reveal unique interactions.
[0743] In addition, protein-protein interactions are confirmed
using two yeast hybrid methodology (Curr Opin Chem Biol. 1999,
3:64). A vector carrying a library of proteins fused to the
activation domain of a transcription factor is introduced into
yeast expressing a 158P1H4-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
colorimetric reporter activity. Specific association with surface
receptors and effector molecules directs one of skill to the mode
of action of 158P1H4, and thus identifies therapeutic, prognostic,
preventative and/or diagnostic targets for cancer. This and similar
assays are also used to identify and screen for small molecules
that interact with 158P1H4.
[0744] When 158P1H4 associates with proteins or small molecules it
is used as a target for diagnostic, prognostic, preventative and
therapeutic purposes.
Example 45
[0745] SSH-Generated Isolation of a cDNA Fragment of the 158P1F4
Gene
[0746] To isolate genes that are over-expressed in bladder cancer
we used the Suppression Subtractive Hybridization (SSH) procedure
using cDNA derived from bladder cancer tissues, including invasive
transitional cell carcinoma. The 158P1F4 SSH cDNA sequence was
derived from a bladder cancer pool minus normal bladder cDNA
subtraction. Included in the driver were also cDNAs derived from
nine other normal tissues. The 158P1F4 cDNA was identified as
highly expressed in the bladder cancer tissue pool, with lower
expression seen in a restricted set of normal tissues.
[0747] The SSH DNA sequence of 213 bp (FIG. 1) has homology to a
chromosome 8q23 Bacterial Artificial Chromosome (BAC) clone
(GenBank accession AP001205).
[0748] Materials and Methods
[0749] Human Tissues
[0750] The bladder cancer and normal tissues were purchased from
several sources, such as the NDRI (Philadelphia, Pa.). mRNA for
some normal tissues were purchased from Clontech, Palo Alto,
Calif.
[0751] RNA Isolation
[0752] Tissues were homogenized in Trizol reagent (Life
Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA.
Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA
Mini and Midi kits. Total and mRNA were quantified by
spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel
electrophoresis.
[0753] Oligonucleotides
[0754] The following HPLC purified oligonucleotides were used.
3 DPNCDN (cDNA synthesis primer): +TL,22 5'TTTTGATCAAGCTT.sub.303'
(SEQ ID NO:729) Adaptor 1: 5'CTAATACGACTCACTATAGGGCTCGAGCGGCC (SEQ
ID NO:730) GCCCGGGCAG3' 3'GGCCCGTCCTAG5' (SEQ ID NO:731) Adaptor 2:
5'GTAATACGACTCACTATAGGGCAGCGTGGTCG (SEQ ID NO:732) CGGCCGAG3'
3'CGGCTCCTAG5' (SEQ ID NO:733) PCR primer 1:
5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO:734) Nested primer (NP)1:
5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO:735) Nested primer (NP)2:
5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO:736)
[0755] Suppression Subtractive Hybridization
[0756] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in bladder cancer. The SSH reaction utilized cDNA from
bladder cancer and normal tissues.
[0757] The gene 158P1F4 sequence was derived from a bladder cancer
pool minus normal bladder cDNA subtraction. The SSH DNA sequence
(FIG. 1) was identified.
[0758] The cDNA derived from of pool of normal bladder tissues was
used as the source of the "driver" cDNA, while the cDNA from a pool
of bladder cancer tissues was used as the source of the "tester"
cDNA. Double stranded cDNAs corresponding to tester and driver
cDNAs were synthesized from 2 .mu.g of poly(A).sup.+ RNA isolated
from the relevant xenograft tissue, as described above, using
CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of
oligonucleotide DPNCDN as primer. First- and second-strand
synthesis were carried out as described in the Kit's user manual
protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The
resulting cDNA was digested with Dpn II for 3 hrs at 37.degree. C.
Digested cDNA was extracted with phenol/chloroform (1:1) and
ethanol precipitated.
[0759] Driver cDNA was generated by combining in a 1:1 ratio Dpn II
digested cDNA from the relevant tissue source (see above) with a
mix of digested cDNAs derived from the nine normal tissues:
stomach, skeletal muscle, lung, brain, liver, kidney, pancreas,
small intestine, and heart.
[0760] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH).
Ligation was terminated with 1 .mu.l of 0.2 M EDTA and heating at
72.degree. C. for 5 min.
[0761] The first hybridization was performed by adding 1.5 .mu.l
(600 ng) of driver cDNA to each of two tubes containing 1.5 .mu.l
(20 ng) Adaptor 1- and Adaptor 2- ligated tester cDNA. In a final
volume of 4 .mu., the samples were overlaid with mineral oil,
denatured in an M J Research thermal cycler at 98.degree. C. for
1.5 minutes, and then were allowed to hybridize for 8 hrs at
68.degree. C. The two hybridizations were then mixed together with
an additional 1 .mu.l of fresh denatured driver cDNA and were
allowed to hybridize overnight at 68.degree. C. The second
hybridization was then diluted in 200 .mu.l of 20 mM Hepes, pH 8.3,
50 mM NaCl, 0.2 mM EDTA, heated at 70.degree. C. for 7 min. and
stored at -20.degree. C.
[0762] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH
[0763] To amplify gene fragments resulting from SSH reactions, two
PCR amplifications were performed. In the primary PCR reaction 1
.mu.l of the diluted final hybridization mix was added to 1 .mu.l
of PCR primer 1 (10 .mu.M), 0.5 .mu.l dNTP mix (10 .mu.M), 2.5
.mu.l 10.times. reaction buffer (CLONTECH) and 0.5 .mu.l 50.times.
Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25
.mu.l. PCR 1 was conducted using the following conditions:
75.degree. C. for 5 min., 94.degree. C. for 25 sec., then 27 cycles
of 94.degree. C. for 10 sec, 66.degree. C. for 30 sec, 72.degree.
C. for 1.5 min. Five separate primary PCR reactions were performed
for each experiment. The products were pooled and diluted 1:10 with
water. For the secondary PCR reaction, 1 .mu.l from the pooled and
diluted primary PCR reaction was added to the same reaction mix as
used for PCR 1, except that primers NP1 and NP2 (10 .mu.M) were
used instead of PCR primer 1. PCR 2 was performed using 10-12
cycles of 94.degree. C. for 10 sec, 68.degree. C. for 30 sec, and
72.degree. C. for 1.5 minutes. The PCR products were analyzed using
2% agarose gel electrophoresis.
[0764] The PCR products were inserted into pCR2.1 using the T/A
vector cloning kit (Invitrogen). Transformed E. coli were subjected
to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in liquid culture
overnight. To identify inserts, PCR amplification was performed on
1 ml of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0765] Bacterial clones were stored in 20% glycerol in a 96 well
format. Plasmid DNA was prepared, sequenced, and subjected to
nucleic acid homology searches of the GenBank, dbest, and NCI-CGAP
databases.
[0766] RT-PCR Expression Analysis
[0767] First strand cDNAs can be generated from 1 .mu.g of mRNA
with oligo (dT)12-18 priming using the Gibco-BRL Superscript
Preamplification system. The manufacturer's protocol was used which
included an incubation for 50 min at 42.degree. C. with reverse
transcriptase followed by RNAse H treatment at 37.degree. C. for 20
min. After completing the reaction, the volume can be increased to
200 .mu.l with water prior to normalization. First strand cDNAs
from 16 different normal human tissues can be obtained from
Clontech.
[0768] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers
5'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: 737) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 738) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl.sub.2,
50 mM KCl, pH8.3) and 1.times.Klentaq DNA polymerase (Clontech).
Five .mu.l of the PCR reaction can be removed at 18, 20, and 22
cycles and used for agarose gel electrophoresis. PCR was performed
using an M J Research thermal cycler under the following
conditions: Initial denaturation can be at 94.degree. C. for 15
sec, followed by a 18, 20, and 22 cycles of 94.degree. C. for 15,
65.degree. C. for 2 min, 72.degree. C. for 5 sec. A final extension
at 72.degree. C. was carried out for 2 min. After agarose gel
electrophoresis, the band intensities of the 283 b.p. .beta.-actin
bands from multiple tissues were compared by visual inspection.
Dilution factors for the first strand cDNAs were calculated to
result in equal .beta.-actin band intensities in all tissues after
22 cycles of PCR. Three rounds of normalization can be required to
achieve equal band intensities in all tissues after 22 cycles of
PCR.
[0769] To determine expression levels of the 158P1F4 gene, 5 .mu.l
of normalized first strand cDNA were analyzed by PCR using 26, and
30 cycles of amplification. Semi-quantitative expression analysis
can be achieved by comparing the PCR products at cycle numbers that
give light band intensities. The primers used for RT-PCR were
designed using the 158P1F4-SSH sequences and are listed below:
4 158P1F4.1 5'-GTCACCGAGCAATGTGATTGAGTT3' (SEQ ID NO:739) 158P1F4.2
5'-GATCCAAATGACCAATCAGGAGA-3' (SEQ ID NO:740)
[0770] A typical RT-PCR expression analysis is shown in FIG. 15.
RT-PCR expression analysis was performed on first strand cDNAs
generated using pools of tissues from multiple samples. The cDNAs
were shown to be normalized using beta-actin PCR. Expression of
158P1F4 was observed in bladder cancer pool.
Example 46
[0771] Chromosomal Mapping of 158P1F4
[0772] Chromosomal localization can implicate genes in disease
pathogenesis. Several chromosome mapping approaches are available
including fluorescent in situ hybridization (FISH), human/hamster
radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics
7:22; Research Genetics, Huntsville Ala.), human-rodent somatic
cell hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI Bethesda, Md.).
[0773] 158P1F4 maps to chromosme 8q23, using 158P1F4 sequence and
the NCBI-BLAST tool:
(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBla-
st.html&&ORG=Hs).
[0774] This is a region of frequent amplification in bladder cancer
(Prat et al., Urology May 2001;57(5):986-92; Muscheck et al.,
Carcinogenesis September2000;21(9):1721-26) and is associated with
rapid tumor cell proliferation in advanced bladder cancer (Tomovska
et al., Int J Oncol June 2001;18(6):1239-44).
Example 47
[0775] Expression analysis of 158P1F4 in Normal Tissues and Patient
Specimens
[0776] Analysis by RT-PCR demonstrates that 158P1F4 expression is
restricted to bladder cancer samples (FIG. 6). First strand cDNA
was prepared from Vital Pool 1 (liver, kidney, and lung), Vital
Pool 2 (stomach, pancreas, colon), and bladder cancer pool.
Normalization was performed by PCR, using primers to actin and
GAPDH. Semi-quantitative PCR, using primers to 158P1F4 was
performed at 26 and 30 cycles of amplification. Expression of
158P1F4 is observed in bladder cancer pool but not in normal
tissues indicating that 158P1F4 serves as a bladder tumor
marker.
[0777] Extensive Northern blot analysis of 158P1F4 in 16 human
normal tissues confirms the expression observed by RT-PCR (FIG. 7).
Even at high exposure length, no expression is detected in the 16
normal tissues tested.
[0778] Northern blot analysis shows that 158P1F4 is expressed in
bladder tumor tissues derived from bladder cancer patients, but not
in normal prostate, baldder and kidney tissues (FIG. 8). This
indicates that 158P1F4 represents a suitable baldder cancer target
for cancer diagnosis and therapy.
Example 48
[0779] Production of Recombinant 158P1F4 in Prokaryotic Systems
[0780] A. In vitro transcription and translation constructs
[0781] pCRII: To generate 158P1F4 sense and anti-sense RNA probes
for RNA in situ investigations, pCRII constructs (Invitrogen,
Carlsbad Calif.) are generated encoding either all or fragments of
the 158P1F4 cDNA. The pCRII vector has Sp6 and T7 promoters
flanking the insert to drive the transcription of 158P1F4 RNA for
use as probes in RNA in situ hybridization experiments. These
probes are used to analyze the cell and tissue expression of
158P1F4 at the RNA level. Transcribed 158P1F4 RNA representing the
cDNA amino acid coding region of the 158P1 F4 gene is used in in
vitro translation systems such as the TnT.TM. Coupled
Reticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize
158P1F4 protein.
[0782] B. Bacterial Constructs
[0783] pGEX Constructs: To generate recombinant 158P1F4 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the 158P1F4 cDNA protein coding sequence
are fused in frame to the GST gene by cloning into pGEX-6P-1 or any
other GST-fusion vector of the pGEX family (Amersham Pharmacia
Biotech, Piscataway, N.J.). These constructs allow controlled
expression of recombinant 15 8P1 F4 protein sequences with GST
fused at the N-terminus and a six histidine epitope at the
C-terminus. The GST and HIS tags permit purification of the
recombinant fusion protein from induced bacteria with the
appropriate affinity matrix and allow recognition of the fusion
protein with anti-GST and HIS antibodies. The six histidine epitope
tag (6.times.His) is generated by adding 6 histidine codons to the
cloning primer at the 3' end of the open reading frame (ORF). A
proteolytic cleavage site, such as the PreScission.TM. recognition
site in pGEX-6P-1, may be employed such that it permits cleavage of
the GST tag from 158PlF4-related protein. The ampicillin resistance
gene and pBR322 origin permits selection and maintenance of the
pGEX plasmids in E. coli. For example, constructs are made
utilizing pGEX-6P-1 such that the whole protein coding region of
158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino
acids from 158P1F4 or analogs thereof are fused at the N-terminus
to GST.
[0784] pMAL Constructs: To generate recombinant 158P1F4 proteins
that are fused to maltose-binding protein (MBP) in bacterial cells,
all or parts of the 158P1F4 cDNA protein coding sequence are fused
in frame to the MBP gene by cloning into the pMAL-c2.times. and
pMAL-p2.times. vectors (New England Biolabs, Beverly, Mass.). These
constructs allow controlled expression of recombinant 158P1F4
protein sequences with MBP fused at the N-terminus and a six
histidine epitope at the C-terminus. The MBP and HIS tags permit
purification of the recombinant protein from induced bacteria with
the appropriate affinity matrix and allow recognition of the fusion
protein with anti-MBP and anti-HIS antibodies. The six histidine
epitope tag is generated by adding the histidine codons to the
3'cloning primer. A Factor Xa recognition site permits cleavage of
the pMAL tag from 158P1F4. The pMAL-c2.times. and pMAL-p2.times.
vectors are optimized to express the recombinant protein in the
cytoplasm or periplasm respectively. Periplasm expression enhances
folding of proteins with disulfide bonds. For example, constructs
are made utilizing pMAL-c2.times. and pMAL-p2.times. vectors such
that the whole protein coding region of 158P1F4 or any 8, 9, 10,
11, 12,13, 14,15, or more contiguous amino acids from 158P1F4 or
analogs thereof are fused at the N-terminus to MBP.
[0785] pET Constructs: To express 1 58P1F4 in bacterial cells, all
or parts of the 158P1F4 cDNA protein coding sequence are cloned
into the pET family of vectors (Novagen, Madison, Wis.). These
vectors allow tightly controlled expression of recombinant 158P1F4
protein in bacteria with and without fusion to proteins that
enhance solubility, such as NusA and thioredoxin (Trx), and epitope
tags, such as 6.times.His and S-Tag.TM. that aid purification and
detection of the recombinant protein. For example, constructs are
made utilizing pET NusA fusion system 43.1 such that the whole
protein coding region of 158P1F4 or any 8, 9, 10, 11, 12,13, 14,15,
or more contiguous amino acids from 158P1F4 or analogs thereof are
fused at the N-terminus to NusA.
[0786] c. Yeast Constructs
[0787] PESC Constructs: To express 158P1F4 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 158P1F4 cDNA protein coding
sequence are cloned into the pESC family of vectors each of which
contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3
(Stratagene, La Jolla, Calif.). These vectors allow controlled
expression from the same plasmid of up to 2 different genes or
cloned sequences containing either Flag.TM. or Myc epitope tags in
the same yeast cell. This system is useful to confirm
protein-protein interactions of 158P1F4. In addition, expression in
yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations, that are found when expressed
in eukaryotic cells. For example, constructs are made utilizing
pESC-HIS such that the whole protein coding region of 158P1F4 or
any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from
158P1F4 or analogs thereof are fused at the carboxyl-terminus to a
6.times.His tag.
[0788] pESP Constructs: To express 158P1F4 in the yeast species
Saccharomyces pombe, all or parts of the 158P1F4 sequence are
cloned into the pESP family of vectors. These vectors allow
controlled high level of expression of a 158P1F4 protein sequence
that is fused at either the amino terminus or at the carboxyl
terminus to GST which aids purification of the recombinant protein.
A Flag.TM. epitope tag allows detection of the recombinant protein
with anti-Flag.TM. antibody. For example, constructs are made
utilizing pESP-1 vector such that the whole protein coding region
of 158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous
amino acids from 158P1F4 or analogs thereof are fused at the
carboxyl-terminus to a 6.times.His tag.
Example 49
[0789] Production of Recombinant 158P1F4 in Eukaryotic Systems
[0790] A. Mammalian Constructs
[0791] To express recombinant 158P1F4 in eukaryotic cells, the full
or partial length 158P1F4 cDNA sequences can be cloned into any one
of a variety of expression vectors known in the art. The constructs
can be transfected into any one of a wide variety of mammalian
cells such as 293T cells. Transfected 293T cell lysates can be
probed with the anti-158P1F4 polyclonal serum, described above.
[0792] pcDNA4/HisMax Constructs: To express 158P1F4 in mammalian
cells, the 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 are cloned into pcDNA4/HisMax
Version A (Invitrogen, Carlsbad, Calif.). Protein expression is
driven from the cytomegalovirus (CMV) promoter and the SP163
translational enhancer. The recombinant protein has Xpress.TM. and
six histidine (6.times.His) epitopes fused to the amino-terminus.
The pcDNA4/HisMax vector also contains the bovine growth hormone
(BGH) polyadenylation signal and transcription termination sequence
to enhance mRNA stability along with the SV40 origin for episomal
replication and simple vector rescue in cell lines expressing the
large T antigen. The Zeocin resistance gene allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and Co1E1 origin permits selection and maintenance
of the plasmid in E. coli. The whole protein coding region of
158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino
acids from 158P1F4 or analogs thereof are expressed in these
constructs.
[0793] pcDNA3.1/MycHis Constructs: To express 158P1F4 in mammalian
cells, the 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 with a consensus Kozak
translation initiation site are cloned into pcDNA3.1/MycHis Version
A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from
the cytomegalovirus (CMV) promoter. The recombinant proteins have
the myc epitope and 6.times.His epitope fused to the
carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the
bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli. The whole protein coding region of
158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino
acids from 158P1F4 or analogs thereof are expressed in these
constructs.
[0794] pcDNA3.1/CT-GFP-TOPO Construct: To express 158P1F4 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, the 158P1F4 ORF or sequences coding for a
stretch of contiguous amino acids of 158P1F4 with a consensus Kozak
translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO
(Invitrogen, Calif.). Protein expression is driven from the
cytomegalovirus (CMV) promoter. The recombinant proteins have the
Green Fluorescent Protein (GFP) fused to the carboxyl-terminus
facilitating non-invasive, in vivo detection and cell biology
studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine
growth hormone (BGH) polyadenylation signal and transcription
termination sequence to enhance mRNA stability along with the SV40
origin for episomal replication and simple vector rescue in cell
lines expressing the large T antigen. The Neomycin resistance gene
allows for selection of mammalian cells that express the protein,
and the ampicillin resistance gene and ColE1 origin permits
selection and maintenance of the plasmid in E. coli. Additional
constructs with an amino-terminal GFP fusion are made in
pcDNA3.1/NT-GFP-TOPO spanning the entire length of the 158P1F4
proteins. The whole protein coding region of 158P1F4 or any 8, 9,
10, 11, 12,13, 14,15, or more contiguous amino acids from 158P1F4
or analogs thereof are expressed in these constructs.
[0795] PAPtag: The 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 are cloned into pAPtag-5
(GenHunter Corp. Nashville, Tenn.). This construct generates an
alkaline phosphatase fusion at the carboxyl-terminus of the 158P1F4
proteins while fusing the IgGic signal sequence to the
amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgGK signal sequence is fused to
the amino-terminus of 158P2H4 proteins. The resulting recombinant
158P1F4 proteins are optimized for secretion into the media of
transfected mammalian cells and can be used to identify proteins
such as ligands or receptors that interact with the 158P1F4
proteins. Protein expression is driven from the CMV promoter and
the recombinant proteins also contain myc and 6.times.His epitopes
fused at the carboxyl-terminus that facilitates detection and
purification. The Zeocin resistance gene present in the vector
allows for selection of mammalian cells expressing the recombinant
protein and the ampicillin resistance gene permits selection of the
plasmid in E. coli. The whole protein coding region of 158P1F4 or
any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from
158P1F4 or analogs thereof are expressed in these constructs.
[0796] ptag5: The 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 are also cloned into pTag-5. This
vector is similar to pAPtag but without the alkaline phosphatase
fusion. This construct generates 158P1F4 proteins with an
amino-terminal IgGK signal sequence and myc and 6.times.His epitope
tags at the carboxyl-terminus that facilitate detection and
affinity purification. The resulting recombinant 158P1F4 proteins
are optimized for secretion into the media of transfected mammalian
cells, and can be used as immunogens or ligands to identify
proteins such as ligands or receptors that interact with the
158P1F4 proteins. Protein expression is driven from the CMV
promoter. The Zeocin resistance gene present in the vector allows
for selection of mammalian cells expressing the protein, and the
ampicillin resistance gene permits selection of the plasmid in E.
coli. The whole protein coding region of 158P1F4 or any 8, 9, 10,
11, 12,13, 14,15, or more contiguous amino acids from 158P1F4 or
analogs thereof are expressed in these constructs.
[0797] PsecFc: The 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 are also cloned into psecFc. The
psecFc vector was assembled by cloning the human immunoglobulin G1
(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,
Calif.). This construct generates an IgG1 Fc fusion at the
carboxyl-terminus of the 158P1F4 proteins, while fusing the IgGK
signal sequence to N-terminus. 158P1F4 fusions utilizing the murine
IgGl Fc region are also used. The resulting recombinant 158P1F4
proteins are optimized for secretion into the media of transfected
mammalian cells, and can be used as immunogens or to identify
proteins such as ligands or receptors that interact with the
158P1F4 protein. Protein expression is driven from the CMV
promoter. The hygromycin resistance gene present in the vector
allows for selection of mammalian cells that express the
recombinant protein, and the ampicillin resistance gene permits
selection of the plasmid in E. coli . The whole protein coding
region of 158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more
contiguous amino acids from 158P1F4 or analogs thereof are
expressed in these constructs.
[0798] pSR.alpha. Constructs: To generate mammalian cell lines that
express 158P1F4 constitutively, the ORF or sequences coding for a
stretch of contiguous amino acids of 158P1F4 are cloned into
pSR.alpha. constructs. Amphotropic and ecotropic retroviruses are
generated by transfection of pSR.alpha. constructs into the
293T-10A1 packaging line or co-transfection of pSR.alpha. and a
helper plasmid (containing deleted packaging sequences) into the
293 cells, respectively. The retrovirus can be used to infect a
variety of mammalian cell lines, resulting in the integration of
the cloned gene, 158P1F4, into the host cell-lines. Protein
expression is driven from a long terminal repeat (LTR). The
Neomycin resistance gene present in the vector allows for selection
of mammalian cells that express the protein, and the ampicillin
resistance gene and ColE1 origin permit selection and maintenance
of the plasmid in E. coli . The retroviral vectors can thereafter
be used for infection and generation of various cell lines using,
for example, SCaBER, NIH 3T3, TsuPr1, 293 or rat-1 cells.
[0799] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
158P1F4 sequences to allow detection using anti-Flag antibodies.
For example, the FLAG.TM. sequence 5' gat tac aag gat gac gac gat
aag 3' is added to cloning primer at the 3' end of the ORF.
Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the fill-length 158P1F4 proteins. The whole protein
coding region of 158P1F4 or any 8, 9, 10, 11, 12,13, 14,15, or more
contiguous amino acids from 158P1F4 or analogs thereof -are
expressed in these constructs.
[0800] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 158P1F4. High virus titer
leading to high level expression of 158P1F4 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. The 158P1F4 coding sequences or fragments thereof are
amplified by PCR and subcloned into the AdEasy shuttle vector
(Stratagene). Recombination and virus packaging are performed
according to the manufacturer's instructions to generate adenoviral
vectors. Alternatively, 158P1F4 coding sequences or fragments
thereof are cloned into the HSV-1 vector (Imgenex) to generate
herpes viral vectors. The viral vectors are thereafter used for
infection of various cell lines such as SCaBER, NIH 3T3, 293 or
rat-1 cells. The whole protein coding region of 158P1F4 or any 8,
9, 10, 11, 12,13, 14,15, or more contiguous amino acids from
158P1F4 or analogs thereof are expressed in these constructs.
[0801] Regulated Expression Systems: To control expression of
158P1F4 in mammalian cells, coding sequences of 158P1F4 are cloned
into regulated mammalian expression systems such as the T-Rex
System (Invitrogen), the GeneSwitch System (Invitrogen) and the
tightly-regulated Ecdysone System (Sratagene). These systems allow
the study of the temporal and concentration dependent effects of
recombinant 158P1F4. These vectors are thereafter used to control
expression of 158P1F4 in various cell lines such as SCaBER, NIH
3T3, 293 or rat-I cells. The whole protein coding region of 158P1F4
or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids
from 158P1F4 or analogs thereof are expressed in these
constructs.
[0802] B. Baculovirus Expression Systems
[0803] To generate recombinant 158P1F4 proteins in a baculovirus
expression system, 158P1F4 ORF or sequences coding for a stretch of
contiguous amino acids of 158P1F4 are cloned into the baculovirus
transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag
at the N-terminus. Specifically, pBlueBac-158P1F4 is co-transfected
with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera
frugiperda) insect cells to generate recombinant baculovirus (see
Invitrogen instruction manual for details). Baculovirus is then
collected from cell supernatant and purified by plaque assay.
[0804] Recombinant 158P1F4 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 158P1F4 protein can be detected using anti-158P1F4 or
anti-His-tag antibody. 158P1F4 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 158P1F4.
[0805] The whole protein coding region of 158P1F4 or any 8, 9, 10,
11, 12,13, 14,15, or more contiguous amino acids from 158P1F4 or
analogs thereof are expressed in these constructs.
Example 50
[0806] Identification of Signal Transduction Pathways
[0807] Many mammalian proteins have been reported to interact with
signaling molecules and to participate in regulating signaling
pathways. (J Neurochem 2001; 76:217-223). Based on the expression
pattern of 158P1F4, it participates in regulating biologically
important signaling cascades. Using immunoprecipitation and Western
blotting techniques, proteins are identified that associate with
158P1H4 and mediate signaling events. Several pathways known to
play a role in cancer biology are regulated by 158P1F4, including
phospholipid pathways such as PI3K, AKT, etc, adhesion and
migration pathways, including FAK, Rho, Rac-1, etc, as well as
mitogenic/survival cascades such as ERK, p38, etc (Cell Growth
Differ. 2000,11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,
19:3003, J. Cell Biol. 1997, 138:913.). Bioinformatic analysis
revealed that 158P1F4 can be phosphorylated by serine/threonine as
well as tyrosine kinases. Phosphorylation of 158P1F4 leads to
activation of one or more of the above listed pathways.
[0808] Using, e.g., Western blotting techniques the ability of
158P1F4 to regulate these pathways is confirmed. Cells expressing
or lacking 158P1F4 are either left untreated or stimulated with
cytokines, growth factors and anti-integrin antibodies. Cell
lysates are analyzed using anti-phospho-specific antibodies (Cell
Signaling, Santa Cruz Biotechnology) in order to detect
phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and
other signaling molecules. When 158P1H4 plays a role in the
regulation of signaling pathways, whether individually or
communally, it is used as a target for diagnostic, prognostic,
preventative and therapeutic purposes.
[0809] To confirm that 158P1F4 directly or indirectly activates
known signal transduction pathways in cells, luciferase (luc) based
transcriptional reporter assays are carried out in cells expressing
individual genes. These transcriptional reporters contain
consensus-binding sites for known transcription factors that lie
downstream of well-characterized signal transduction pathways. The
reporters and examples of these associated transcription factors,
signal transduction pathways, and activation stimuli are listed
below.
[0810] 7. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0811] 8. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0812] 9. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0813] 10. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0814] 11. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0815] 12. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0816] Gene-mediated effects can be assayed in cells showing MRNA
expression. Luciferase reporter plasmids can be introduced by
lipid-mediated transfection (TFX-50, Promega). Luciferase activity,
an indicator of relative transcriptional activity, is measured by
incubation of cell extracts with luciferin substrate and
luminescence of the reaction is monitored in a luminometer.
[0817] Signaling pathways activated by 158P1F4 are mapped and used
for the identification and validation of therapeutic targets. When
158P1F4 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and therapeutic purposes.
Example 51
[0818] Involvement in Tumor Progression
[0819] The 158P1F4 gene contributes to the growth of cancer cells.
The role of 158P1F4 in tumor growth is investigated in a variety of
primary and transfected cell lines including prostate, colon,
bladder and kidney cell lines as well as NIH 3T3 cells engineered
to stably express 15 8P1 F4. Parental cells lacking 158P1F4 and
cells expressing 158P1F4 are evaluated for cell growth using a
well-documented proliferation assay (Fraser S P, Grimes J A,
Djamgoz M B. Prostate. 2000;44:61, Johnson D E, Ochieng J, Evans S
L. Anticancer Drugs. 1996, 7:288).
[0820] To determine the role of 158P1F4 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH3T3 cells lacking 158P1F4 are compared to NHI-3T3 cells
expressin 158P1H4, using a soft agar assay under stringent and more
permissive conditions (Song Z. et al. Cancer Res. 2000,
60:6730).
[0821] To determine the role of 158P1F4 in invasion and metastasis
of cancer cells, a well-established assay is used, e.g., a
Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999,
59:6010). Control cells, including prostate, colon, bladder and
kidney cell lines lacking 158P1F4 are compared to cells expressing
158P1F4. Cells are loaded with the fluorescent dye, calcein, and
plated in the top well of the Transwell insert coated with a
basement membrane analog. Invasion is determined by fluorescence of
cells in the lower chamber relative to the fluorescence of the
entire cell population.
[0822] 158P1F4 also plays a role in cell cycle and apoptosis.
Parental cells and cells expressing 158P1F4 are compared for
differences in cell cycle regulation using a well-established BrdU
assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short,
cells are grown under both optimal (full serum) and limiting (low
serum) conditions are labeled with BrdU and stained with anti-BrdU
Ab and propidium iodide. Cells are analyzed for entry into the G1,
S, and G2M phases of the cell cycle. Alternatively, the effect of
stress on apoptosis is evaluated in control parental cells and
cells expressing 158P1F4, including normal and tumor bladder cells.
Engineered and parental cells are treated with various
chemotherapeutic agents, such as paclitaxel, gemcitabine, etc, and
protein synthesis inhibitors, such as cycloheximide. Cells are
stained with annexin V-FITC and cell death is measured by FACS
analysis. The modulation of cell death by 158P1F4 plays a critical
role in regulating tumor progression and tumor load.
[0823] When 158P1F4 plays a role in cell growth, transformation,
invasion and/or apoptosis, it is used as a target for diagnostic,
prognostic, preventative and therapeutic purposes.
Example 52
[0824] In Vivo Assay for 158P1F4 Tumor Growth Promotion
[0825] The effect of the 158P1F4 protein on tumor cell growth is
confirmed in vivo by gene overexpression in bladder cancer cells.
For example, SCID mice can be injected SQ on each flank with
1.times.10.sup.6 bladder cancer cells (such as SCaBER, UM-UC-3,
HT1376, RT4, T24, TCC-SUP, J82 and SW780 cells) containing tkNeo
empty vector or 158P1F4.
[0826] At least two strategies may be used: (1) Constitutive
158P1H4 expression under regulation of a a constitutive promoter
such as those obtained from the genomes of viruses such as polyoma
virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), or from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, provided
such promoters are compatible with the host cell systems. (2)
Regulated expression under control of an inducible vector system,
such as ecdysone, tet, etc., can be used provided such promoters
are compatible with the host cell systems. Tumor volume is then
monitored by the appearance of palpable tumors. Tumor growth is
followed over time and it is determine that 158P1F4-expressing
cells grow at a faster rate and tumors produced by
158P1F4-expressing cells demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs). Additionally, mice can
be implanted with the same cells orthotopically and the effect of
158P1F4 on local growth in the bladder and/or on the ability of the
cells to metastasize, specifically to lungs or lymph nodes is
seen(Fu, X., et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang,
S., et al., Anticancer Res., 1997. 17: p.3239-3242; Peralta, E. A.,
et al., J. Urol., 1999.162: p. 1806-1811).
[0827] Furthermore, this assay is useful to determine the 158P1F4
inhibitory effect of candidate therapeutic compositions, such as
for example, 158P1F4 antibodies or intrabodies, and 158P1F4
antisense molecules or ribozymes.
Example 54
[0828] Homology Comparison of 158P1F4 to Known Sequences
[0829] The 158P1F4 gene shows some homology to a hypothetical human
protein (gi 14742939) and partially aligns with a human potassium
channel (gi 14784073) (95% identity over 23 bp)
[http://www.ncbi.nlm.nih.gov/entr- ez], indicating that the full
length gene product may correspond to a known family of
proteins.
[0830] gi.vertline.14742939.vertline.ref.vertline.XM
050239.1.vertline.Homo sapiens hypothetical gene . . . 42 0.15
[0831]
gi.vertline.11875312.vertline.gb.vertline.AC008267.6.vertline.AC008-
267 Homo sapiens BAC clone G . . . 42 0.15
[0832]
gi.vertline.11136704.vertline.jb.vertline.AC008892.5.vertline.AC008-
892 Homo sapiens chromosome ... 42 0.15
[0833]
gi.vertline.14916145.vertline.gb.vertline.AC011459.4.vertline.AC011-
459 Homo sapiens chromosome ... 38 2.3
[0834] gi.vertline.14784073.vertline.ref.vertline.XM
035782.1.vertline. Homo sapiens potassium channel . . . 38 2.3
[0835] gi.vertline.14784071.vertline.ref.vertline.XM
035781.1.vertline. Homo sapiens potassium channel . . . 38 2.3
[0836] gi.vertline.14784069.vertline.ref.vertline.XM
006515.4.vertline. Homo sapiens potassium channel . . . 38 2.3
Example 55
[0837] Splice Variants of 158P1H4
[0838] Splice variants are also called alternative transcripts.
When a gene is transcribed from genomic DNA, the initial RNA is
generally spliced to produce functional MRNA, which has only exons
and is used for translation into an amino acid sequence.
Accordingly, a given gene can have zero to many alternatively
spliced mRNA products. Alternative transcripts each have a unique
exon makeup, and can have different coding and/or non-coding (5' or
3' end) portions, from the original transcript. Alternative
transcripts can code for similar proteins with the same or a
similar function or may encode proteins with different functions,
and may be expressed in the same tissue at the same time, or at
different tissue at different times, proteins encoded by
alternative transcripts can have similar or different cellular or
extracellular localizations, e.g., be secreted.
[0839] Splice variants are identified by a variety of art-accepted
methods. For example, splice variants are identified by use of EST
data. First, all human ESTs were grouped into clusters which show
direct or indirect identity with each other. Second, ESTs in the
same cluster were further grouped into sub-clusters and assembled
into a consensus sequence. The starting gene is compared to the
consensus sequence(s). Each consensus sequence is a potential
splice variant for that gene (see, e.g.,
http://www.doubletwist.com/products/c11 agentsOverview.jhtml). Even
when a variant is identified that is not a full-length clone, that
portion of the variant is very useful for antigen generation and
for further cloning of the full-length splice variant, using
techniques known in the art.
[0840] Moreover, computer programs are available in the art that
identify splice variants based on genomic sequences. Genomic-based
variant identification programs include FgenesH (A. Salamov and V.
Solovyev, "Ab initio gene finding in Drosophila genomic DNA,"
Genome Research. April 2000;10(4):516-22); Grail
(http://combio.ornl.gov/Grail-bin/EmptyGrailFor- m) and GenScan
(http://genes.mit.edu/GENSCAN.html). For a general discussion of
splce variant identification protocols see., e.g., Southan C., "A
genomic perspective on human proteases," FEBS Lett. Jun. 8,
2001;498(2-3):214-8; de Souza S J, et al., "Identification of human
chromosome 22 transcribed sequences with ORF expressed sequence
tags," Proc. Natl Acad Sci U S A. Nov. 7, 2000;97(23): 12690-3.
[0841] For variants identified by the EST-based method, Table XXI
shows the nucleotide sequences of the splice variants. Table XXII
shows the alignment of the splice variants with the 158P1H4 nucleic
acid sequence. Table XXIII displays the single longest alignment of
an amino acid sequence encoded by a splice variant, out of all six
potential reading frames with 158P1H4. Thus, for each splice
variant, a variant's reading frame that encodes the longest single
contiguous peptide homology between 158P1H4 and the variant is the
proper reading frame orientation for the variant. Due to the
possibility of sequencing errors in EST or genomic data, other
peptides in the relevant reading frame orientation (5' to 3' or 3'
to 5') can also be encoded by the variant. Table XXIV lays out all
three frame shifted amino acid translations of the splice variant
for the identifed reading frame orientation. Tables XXI through
XXIV are set forth herein on a variant-by-variant basis.
[0842] For variants identified by any one of the genomic
sequence-based methods, Table XXI shows the nucleotide sequences of
the splice variant. Table XXII shows the alignment of the splice
variant with the 158P1H4 nucleic acid sequence. Table XXIII
displays the alignment of amino acid sequence of the predicted
transcripts with 158P1H4. The genomic-based computer programs
predict a transcript from genomic sequence, and not only predict
exons but also set open reading frame as the first forward open
reading frame. The predicted transcript does not contain 5' or 3'
untranslated region (UTR). It starts with ATG and ends with a stop
codon, TAG, TGA or TAA. In case the transcript is predicted on the
reverse strand of the genomic sequence, the sequence of the
transcript is reverse-complemented to the genomic sequence of the
exons. Thus, the genomic-based programs provides the correct
transcript sequence, with 5' to 3' orientation and +1 as the open
reading frame. However, due to the possibility of inaccurate
prediction of exons or the possibility of sequencing errors in
genomic data, other peptides in other forward open reading frame
can also be encoded by the variant. Table XXIV lays out all amino
acid translations of the splice variant in each of the three
forward reading frames. Tables XXI through XXIV are set forth
herein on a variant-by-variant basis.
[0843] To further confirm the parameters of a splice variant, a
variety of techniques are available in the art, such as proteomic
validation, PCR-based validation, and 5' RACE validation, etc. (see
e.g., Proteomic Validation: Brennan S O, Fellowes A P, George P M.;
"Albumin banks peninsula: a new termination variant characterised
by electrospray mass spectrometry." Biochim Biophys Acta. 1999 Aug
17;1433(1-2):321-6; Ferranti P, et al., "Differential splicing of
pre-messenger RNA produces multiple forms of mature caprine
alpha(s1)-casein." Eur J Biochem. Oct 1, 1997;249(1):1-7; PCR-based
Validation: Wellmann S, et al., "Specific reverse transcription-PCR
quantification of vascular endothelial growth factor (VEGF) splice
variants by LightCycler technology." Clin Chem. April
2001;47(4):654-60; Jia HP, et al., Discovery of new human
beta-defensins using a genomics-based approach," Gene. Jan. 24,
2001;263(1-2):21 1-8; PCR-based and 5' RACE Validation: Brigle KE,
et al., "Organization of the murine reduced folate carrier gene and
identification of variant splice forms," Biochim Biophys Acta. Aug.
7, 1997; 1353(2): 191-8.
[0844] It is known in the art that genomic regions are upregulated
in cancers. When the genomic region to which 158P1H4 maps is
upregulated in a particular cancer, the splice variants of 158P1H4
are upregulated as well. Disclosed herein is that 158P1H4 has a
particular expression profile. Splice variants of 158P1H4 that are
structurally and/or functionally similar to 158P1H4 share this
expression pattern, thus serving as tumor-associated
markers/antigens.
[0845] Using gene-prediction approaches discussed above, we
identified the two splice variants detailed in Table XXIa through
Table XXIVa (splice variant 1); Table XXIb through Table XXIVb
(splice variant 2).
Example 56
[0846] Expression Analysis of 158P1H4 Splice Variants in Normal
Tissues and Patient Tumor Specimens
[0847] In order to assay for expression of 158P1H4 splice variants
in human tissues, normal tissue Northern blots (FIG. 18C) were
hybridized using 158P4B5 probe 1 which comprises the SSH sequence
of 158P4B5 (FIG. 18B). 158P4B5 probe 1 contains 92 base pairs of
sequences present in splice varia and 2 which are defined in FIGS.
18A and 18B. The results show that 158P4B5 probe 1 detects
expression of multiple size transcripts in a tissue specific
pattern. Most prominent transcripts of 2 kb and 1 kb are detected
mostly in heart and skeletal muscle. Lower expression levels are
detected in other tissues.
[0848] 158P4B5 probe 1 was also used to assay for expression of
158P1H4 splice variants in bladder cancer patient specimens (FIG.
18D). RNA was extracted from normal bladder (Nb), bladder tumors
(T) and their matched normal adjacent tissue (NAT) isolated from
bladder cancer patients. Northern blots with 10 .mu.g of total
RNA/lane were hybridized with 158P4B5 probe 1 defined in FIGS. 18A
and 18B. Size standards in kilobases (kb) are indicated on the
side. The arrow indicates transcripts similar in size to the
transcript detected using 158P1H4 probe. The results show
expression of multiple transcripts in bladder tumors and normal
tissues. In particular, an approximately 4.0 kb transcript is
detected in 2 of 4 bladder tumors but not in normal tissues. These
results indicate that different size transcripts, which share exons
with 158P1H4, are expressed in bladder cancer patient specimens.
These can be defined as splice variants of 158P1H4.
[0849] Expression of 158P4B5 in bladder cancer patient specimens
was also detected using 158P4B5 probe 2 (FIG. 18E). 158P4B5 probe 2
overlaps by 64 bp with sequences of splice variants 1 and 2 (FIGS.
18A and 18B). RNA was extracted from normal bladder (Nb), bladder
tumors (T) and their matched normal adjacent tissues (NAT) isolated
from bladder cancer patients. Northern blots with 10 .mu.g of total
RNA/lane were hybridized with 158P4B5 probe 2 defined in FIG. 18A
and FIG. 18B. The results show expression of one predominant
2.4-4.4 kb transcript in bladder cancer tissues, and low expression
of a larger transcript greater than 7.5 kb in size.
[0850] FIG. 19A provides a schematic and alignment of splice
variant 1 to 158P1H4. Horizontal wave hatching are regions of
158P1H4 not present in either splice variant. Depicted in black are
regions of 158P1H4 present in the variant. The amino acids present
in each region are numbered according to their sequence in 158P1H4.
Depicted with diagonal hatching is a region specific to splice
variant 1 indicative of amino acids arising from an alternative
exon(s) not utilized in 158P1H4 or splice variant 2. Depicted with
horizontal hatching is a region specific to both splice variant 1
and splice variant 2 indicative of amino acids arising from an
alternative exon(s) not utilized in 158P1H4 but utilized in both
variants. This region, combined with the immediate 6 amino-terminal
amino acids arising from sequence present in 158P1H4, encodes a
putative transmembrane domain, determined from sequence analysis
using the TMpred transmembrane prediction algorithm of Hofmann and
Stoffel (K. Hofmann, W. Stoffel. TMBASE-A database of membrane
spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993)
accessed on the ExPasy molecular biology server
(http://www.expasy.ch/tools/). Asterisks indicate single amino acid
deletions in splice variant 1 compared to the 158P1H4 sequence
(amino acids 175 and 204).
[0851] FIG. 19B provides a schematic and alignment of splice
variant 2 to 158P1H4. Depicted in black are regions of 158P1H4
present in the variant. Depicted with diamond hatching are regions
specific to splice variant 2 indicative of amino acids arising from
alternative exons not utilized in 158P1H4 or splice variant 1.
Depicted with horizontal hatching is the region present in splice
variant 1 and splice variant 2 but not 158P1H4. The putative
transmembrane domain is also indicated.
[0852] The expression of several transcripts detected using 158P4B5
probe 1 and probe 2 indicate that 158P1H4 splice variants are
expressed in bladder cancer patient specimens and that these
represent suitable cancer targets for cancer diagnosis and
therapy.
[0853] The present invention is not to be limited in scope by the
embodiments disclosed herein, which are intended as single
illustrations of individual aspects of the invention, and any that
are functionally equivalent are within the scope of the invention.
Various modifications to the models and methods of the invention,
in addition to those described herein, will become apparent to
those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall within the scope of
the invention. Such modifications or other embodiments can be
practiced without departing from the true scope and spirit of the
invention.
[0854] All documents and publications recited herein are hereby
incorporated in their entirety as if fully set forth.
5TABLE I Tissues that Express 158P1H4 When Malignant Bladder
[0855]
6TABLE II AMINO ACID ABBREVIATIONS SINGLE THREE LETTER LETTER FULL
NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine
C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln
glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr
threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine
D Asp aspartic acid E Glu glutamic acid G Gly glycine
[0856]
7TABLE III AMINO ACID SUBSTITUTION MATRIX Adapted from the GCG
Software 9.0 BLOSUM62 amino acid substitution matrix (block
substitution matrix). The higher the value, the more likely a
substitution is found in related, natural proteins. (See URL
www.ikp.unibe.ch/manual/blosum62.html) A C D E F G H I K L M N P Q
R S T V W Y . 4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2
A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C 6 2 -3
-1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D 5 -3 -2 0 -3 1 -3 -2 0
-1 2 0 0 -1 -2 -3 -2 E 6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F
6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G 8 -3 -1 -3 -2 1 -2 0 0
-1 -2 -3 -2 2 H 4 -3 2 1 -3 -3 -3 -3 -2 -1 3 -3 -1 I 5 -2 -1 0 -1 1
2 0 -1 -2 -3 -2 K 4 2 -3 -3 -2 -2 -2 -1 1 -2 -1 L 5 -2 -2 0 -1 -1
-1 1 -1 -1 M 6 -2 0 0 1 0 -3 -4 -2 N 7 -1 -2 -1 -1 -2 -4 -3 P 5 1 0
-1 -2 -2 -1 Q 5 -1 -1 -3 -3 -2 R 4 1 -2 -3 -2 S 5 0 -2 -2 T 4 -3 -1
V 11 2 W 7 Y
[0857]
8 TABLE IV A POSITION POSITION 2 POSITION 3 C Terminus (Primary
Anchor) (Primary Anchor) (Primary Anchor) SUPER- MOTIFS A1 TILVMS
FWY A2 LIVMATQ IVMATL A3 VSMATLI RK A24 VFWIVLMT FIYWLM B7 P
VILFMWYA B27 RHK FYLWMIVA B44 ED FWYLIMVA B58 ATS FWYLIVMA B62
QLIVMP FWYMIVLA MOTIFS A1 TSM Y A1 DEAS Y A2.1 LMVQIAT VLIMAT A3
LMVISATFCGD KYRHFA A11 VTMLISAGNCDF KRYH A24 YFWM FLIW A*3101
MVTALIS RK A*3301 MVALFIST RK A*6801 AVTMSLI RK B*0702 P LMFWYAIV
B*3501 P LMFWYIVA B51 P LIVFWYAM B*5301 P IMFWYALV B*5401 P
ATIVLMFWY Bolded residues are preferred, italicized residues are
less preferred: A peptide is considered motif-bearing if it has
primary anchors at each primary anchor position for a motif or
supermotif as specified in the above table.
[0858]
9TABLE IV (B) HLA CLASS II SUPERMOTIF 1 6 9 W, F, Y, V, .I , L A,
V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y
[0859]
10TABLE IV C MOTIFS 1.degree. anchor 1 2 3 4 5 1.degree. anchor 6 7
8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE
DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD
GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G
GRD N G DR3 MOTIFS 1.degree. anchor 1 2 3 1.degree. anchor 4 5
1.degree. anchor 6 motif a LIVMFY D preferred motif b LIVMFAY
DNQEST KRH preferred DR MFLIVWY VMSTACPLI Supermotif Italicized
residues indicate less preferred or "tolerated" residues.
[0860]
11TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS POSITION: 1 2
3 4 5 6 7 8 C-terminus A1 1 1 .degree. Anchor TILVMS 2 1 .degree.
Anchor FWY A2 3 1 .degree. Anchor LIVMATQ 4 1 .degree. Anchor
LIVMAT A3 preferred 5 1 .degree. Anchor VSMATLI YFW (4/5) YFW (3/5)
YFW (4/5) P (4/5) 6 1 .degree. Anchor RK deleterious DE (3/5); DE P
(5/5) (4/5) A24 7 1 .degree. Anchor YFWIVLMT 8 1 .degree. Anchor
FIYWLM B7 preferred FWY (5/5) LIVM (3/5) 9 1 .degree. Anchor P FWY
(4/5) FWY (3/5) 10 1 .degree. Anchor VILFMWYA deleterious DE (3/5);
DE G QN DE P(5/5); (3/5) (4/5) (4/5) (4/5) G(4/5); A(3/5); QN(3/5)
B27 11 1 .degree. Anchor RHK 12 1 .degree. Anchor FYLWMIVA B44 13 1
.degree. Anchor ED 14 1 .degree. Anchor FWYLIMVA B58 15 1 .degree.
Anchor ATS 16 1 .degree. Anchor FWYLIVMA B62 17 1 .degree. Anchor
QLIVMP 18 1 .degree. Anchor FWYMIVLA
[0861]
12TABLE IV (E) HLA Class I Motifs 9 or POSITION: 1 2 3 4 5 6 7 8
C-terminus C-terminus A1 9-mer preferred GFY W 19 1 .degree. Anchor
STM DEA YFW P DEQN YFW 20 1 .degree. Anchor Y deleterious DE
RHKLIVMP A G A A1 9-mer preferred GRHK ASTCLIVM 21 1 .degree.
Anchor DEAS GSTC ASTC LIVM DE 22 1 .degree. Anchor Y deleterious A
RHKDEPY DE PQN RHK PG GP FW A1 10-mer preferred YFW 23 1 .degree.
Anchor STM DEAQN A YFWQN PASTC GDE P 24 1 .degree. Anchor Y
deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A A1 10-mer preferred
YFW STCLIVM 25 1 .degree. Anchor DEAS A YFW PG G YFW 26 1 .degree.
Anchor Y deletetious RHK RHKDEPY P G PRHK QN FW A2.1 9-mer
preferred YFW 27 1 .degree. Anchor LMIVQAT YFW STC YFW A P 28 1
.degree. Anchor VLIMAT deleterious DEP DERKH RKH DERKH A2.1 10-mer
preferred AYFW 29 1 .degree. Anchor LMIVQAT LVIM G G FYWL VIM 30 1
.degree. Anchor VLIMAT deleterious DEP DE RKHA P RKH DERKLH RKH A3
preferred RHK 31 1 .degree. Anchor LMVISATFCGD YFW PRHKK YFW A YFW
P 32 1 .degree. Anchor KYRHFA deleterious DEP DE A11 preferred A 33
1 .degree. Anchor VTLMISAGNCDF YFW YFW A YFW YFW P 34 1 .degree.
Anchor KRYH deleterious DEP A G A24 9-mer preferred YFWRHK 35 1
.degree. Anchor YFWM STC YFW YFW 36 1 .degree. Anchor FLIW
deleterious DEG DE G QNP DERH G AQN K A24 10-mer preferred 37 1
.degree. Anchor YFWM P YFWP P 38 1 .degree. Anchor FLIW deleterious
GDE QN RHK DE A QN DEA A3101 preferred RHK 39 1 .degree. Anchor
MVTALIS YFW P YFW YFW AP 40 1 .degree. Anchor RK deleterious DEP DE
ADE DE DE DE A3301 preferred 41 1 .degree. Anchor MVALFIST YFW AYFW
42 1 .degree. Anchor RK deleterious GP DE A6801 preferred YFWSTC 43
1 .degree. Anchor AVTMSLI YFWLIV M YFW P 44 1 .degree. Anchor RK
deleterious GP DEG RHK A B0702 preferred RHKFW Y 45 1 .degree.
Anchor P RHK RHK RHK RHK PA 46 1 .degree. Anchor LMFWYAIV
deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV M 47 1
.degree. Anchor P FWY 48 1 .degree. Anchor LMFWYIVA deleterious AGP
G G B51 preferred LIVMFW Y 49 1 .degree. Anchor P FWY STC FWY G FWY
50 1 .degree. Anchor LIVFWYAM deleterious AGPDER HKSTC DE G DEQN
GDE B5301 preferred LIVMFW Y 51 1 .degree. Anchor P FWY STC FWY
LIVMFWY FWY 52 1 .degree. Anchor IMFWYALV deleterious AGPQN G RHKQN
DE B5401 preferred FWY 53 1 .degree. Anchor P FWYL IVM LIVM ALIVM
FWYAP 54 1 .degree. Anchor ATIVLMFWY deleterious GPQNDE GDES TC
RHKDE DE QNDGE DE Italicized residues indicate less preferred or
"tolerated" residues. The information in this Table is specified
for 9-mers unless otherwise specified.
[0862]
13TABLE V HLA Peptide Scoring Results - 158P1H4 - A1, 9-mers Start
Subsequence Residue Score (Estimate of Half Time of Disassociation
of a Rank Position Listing a Molecule Containing This Subsequence)
Seq.ID# 1 273 QLDPCTCDY 250.000 1. 2 13 RSDALGGRY 37.500 2. 3 27
HLDGFLFCR 25.000 3. 4 391 QIEVPEQSK 18.000 4. 5 87 TMDPNVLRS 12.500
5. 6 55 CLPPFPPKY 10.000 6. 7 329 LLDTDGPQR 10.000 7. 8 259
FLELAREVR 9.000 8. 9 384 AAENTEMQI 4.500 9. 10 98 FVEFLKLAQ 4.500
10. 11 394 VPEQSKSKK 4.500 11. 12 263 AREVRHYGY 4.500 12. 13 54
NCLPPFPPK 4.000 13. 14 220 AVDLLYMQA 2.500 14. 15 178 KLADFELPY
2.500 15. 16 209 SLDSVLMDC 2.500 16. 17 44 WNEQLRRVF 2.250 17. 18
193 EVENCKVGL 1.800 18. 19 349 YSEDSCWQW 1.350 19. 20 191 SSEVENCKV
1.350 20. 21 331 DTDGPQRTL 1.250 21. 22 5 FCIPVSQQR 1.000 22. 23 16
ALGGRYVLY 1.000 23. 24 366 FLLSSCLKK 1.000 24. 25 179 LADFELPYV
1.000 25. 26 161 GLFLIRFGK 1.000 26. 27 78 QLEQYLQNV 0.900 27. 28
342 NLELRFQYS 0.900 28. 29 130 RIEIITSDT 0.900 29. 30 142 VLEVVSHKI
0.900 30. 31 94 RSDVFVEFL 0.750 31. 32 305 DSQTQDIVF 0.750 32. 33
135 TSDTAERVL 0.750 33. 34 341 QNLELRFQY 0.625 34. 35 278 TCDYPESGS
0.500 35. 36 108 NTFDIATKK 0.500 36. 37 86 VTMDPNVLR 0.500 37. 38
374 KMISEKMVK 0.500 38. 39 25 SVHLDGFLF 0.500 49. 40 71 MADERRDQL
0.500 40. 41 251 QKEDSQTKF 0.450 41. 42 414 SSFLSRKSK 0.300 42. 43
190 GSSEVENCK 0.300 43. 44 151 GLCRELLGY 0.250 44. 45 67 MTTAMADER
0.250 45. 46 301 ITLPDSQTQ 0.250 46. 47 68 TTAMADERR 0.250 47. 48
350 SEDSCWQWF 0.250 48. 49 294 NNEISCCIT 0.225 49. 50 387 NTEMQIEVP
0.225 50.
[0863]
14TABLE VI HLA Peptide Scoring Results - 158P1H4 - A1, 10-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 94 RSDVFVEFLK 75.000 51. 2 349 YSEDSGWQWF
13.500 52. 3 394 VPEQSKSKKY 11.250 53. 4 54 NCLPPFPPKY 10.000 54. 5
193 EVENCKVGLR 9.000 55. 6 55 CLPPFPPKYY 5.000 56. 7 209 SLDSVLMDCR
5.000 57. 8 308 TQDIVFQMSR 3.750 58. 9 98 FVEFLKLAQL 1.800 59. 10
168 GKEGKLSVVK 1.800 60. 11 259 FLELAREVRH 1.800 61. 12 352
DSCWQWFVLY 1.500 62. 13 229 IQDIEKGWAK 1.500 63. 14 191 SSEVENCKVG
1.350 64. 15 410 QKDYSSFLSR 1.250 65. 16 331 DTDGPQRTLN 1.250 66.
17 109 TFDIATKKAY 1.250 67. 18 294 NNEISCCITL 1.125 68. 19 15
DALGGRYVLY 1.000 69. 20 179 LADFELPYVS 1.000 70. 21 246 KLEAFQKEDS
0.900 71. 22 130 RIEIITSDTA 0.900 72. 23 391 QIEVPEQSKS 0.900 73.
24 78 QLEQYLQNVT 0.900 74. 25 142 VLEVVSHKIG 0.900 75. 26 13
RSDALGGRYV 0.750 76. 27 24 YSVHLDGFLF 0.750 77. 28 376 ISEKMVKLAA
0.675 78. 29 150 IGLCRELLGY 0.625 79. 30 278 TCDYPESGSG 0.500 80.
31 118 YLDIFLPNEQ 0.500 81. 32 225 YMQAIQDIEK 0.500 82. 33 27
HLDGFLFCRV 0.500 83. 34 404 HIQQSQQKDY 0.500 84. 35 329 LLDTDGPQRT
0.500 85. 36 214 LMDCRVAVDL 0.500 86. 37 145 VVSHKIGLCR 0.500 87.
38 427 KDDCVFGNIK 0.500 88. 39 273 QLDPCTCDYP 0.500 89. 40 220
AVDLLYMQAI 0.500 90. 41 338 TLNQNLELRF 0.500 91. 42 281 YPESGSGAVL
0.450 92. 43 44 WNEQLRRVFG 0.450 93. 44 387 NTEMQIEVPE 0.450 94. 45
106 QLNTFDIATK 0.400 95. 46 340 NQNLELRFQY 0.375 96. 47 413
YSSFLSRKSK 0.300 97. 48 173 LSVVKKLADF 0.300 98. 49 29 DGFLFCRVRY
0.250 99. 50 337 RTLNQNLELR 0.250 100.
[0864]
15TABLE VII HLA Peptide Scoring Results - 158P1H4 - A2, 9-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 213 VLMDCRVAV 1793.677 101. 2 156 LLGYFGLFL
759.006 102. 3 205 YMAPSLDSV 260.413 103. 4 91 NVLRSDVFV 123.846
104. 5 359 VIYTKQAFL 87.885 105. 6 198 KVGLRKWYM 62.760 106. 7 375
MISEKMVKL 47.088 107. 8 106 QLNTFDIAT 28.318 108. 9 186 YVSLGSSEV
27.995 109. 10 64 YLAMTTAMA 22.853 110. 11 271 YLQLDPCTC 22.853
111. 12 367 LLSSCLKKM 19.425 112. 13 289 VLSVGNNEI 17.736 113. 14
172 KLSVVKKLA 17.388 114. 15 382 KLAAENTEM 17.388 115. 16 154
RELLGYFGL 13.371 116. 17 321 WQVTFLGTL 11.320 117. 18 40 QLHGWNEQL
10.468 118. 19 78 QLEQYLQNV 10.238 119. 20 179 LADFELPYV 8.310 120.
21 99 VEFLKLAQL 6.009 121. 22 218 RVAVDLLYM 5.499 122. 23 149
KIGLCRELL 5.038 123. 24 157 LGYFGLFLI 4.942 124. 25 209 SLDSVLMDC
4.571 125. 26 178 KLADFELPY 4.064 126. 27 363 KQAFLLSSC 4.055 127.
28 307 QTQDIVFQM 3.774 128. 29 134 ITSDTAERV 3.299 129. 30 31
FLFCRVRYS 3.209 130. 31 252 KEDSQTKFL 3.198 131. 32 1 MKMHFCIPV
2.309 132. 33 255 SQTKFLELA 2.157 133. 34 322 QVTFLGTLL 1.869 134.
35 409 QQKDYSSFL 1.709 135. 36 310 DIVFQMSRV 1.650 136. 37 104
LAQLNTFDI 1.429 137. 38 94 RSDVFVEFL 1.314 138. 39 163 FLIRFGKEG
1.303 139. 40 373 KKMISEKMV 1.251 140. 41 111 DIATKKAYL 1.212 141.
42 101 FLKLAQLNT 1.200 142. 43 142 VLEVVSHKI 1.135 143. 44 337
RTLNQNLEL 1.098 144. 45 364 QAFLLSSCL 1.098 145. 46 284 SGSGAVLSV
1.044 146. 47 300 CITLPDSQT 1.025 147. 48 96 DVFVEFLKL 0.986 148.
49 234 KGWAKPTQA 0.942 149. 50 26 VHLDGFLFC 0.930 150.
[0865]
16TABLE VIII HLA Peptide Scoring Results - 158P1H4 - A2, 10-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 178 KLADFELPYV 12625.601 151. 2 314
QMSRVKCWQV 376.740 152. 3 156 LLGYFGLFLI 236.595 153. 4 103
KLAQLNTFDI 211.786 154. 5 366 FLLSSCLKKM 194.477 155. 6 223
LLYMQAIQDI 130.535 156. 7 374 KMISEKMVKL 124.199 157. 8 155
ELLGYFGLFL 123.897 158. 9 359 VIYTKQAFLL 92.679 159. 10 122
FLPNEQSIRI 47.991 160. 11 416 FLSRKSKIKI 47.991 161. 12 358
FVIYTKQAFL 47.291 162. 13 302 TLPDSQTQDI 42.774 163. 14 70
AMADERRDQL 30.996 164. 15 205 YMAPSLDSVL 29.098 165. 16 212
SVLMDCRVAV 22.517 166. 17 350 SEDSCWQWFV 22.315 167. 18 353
SCWQWFVIYT 19.408 168. 19 77 DQLEQYLQNV 18.453 169. 20 367
LLSSCLKKMI 17.736 170. 21 141 RVLEVVSHKI 13.848 171. 22 83
LQNVTMDPNV 11.988 172. 23 408 SQQKDYSSFL 11.913 173. 24 375
MISEKMVKLA 10.951 174. 25 105 AQLNTFDIAT 10.623 175. 26 214
LMDCRVAVDL 10.491 176. 27 321 WQVTFLGTLL 9.963 177. 28 379
KMVKLAAENT 9.230 178. 29 133 IITSDTAERV 7.966 179. 30 363
KQAFLLSSCL 7.581 180. 31 306 SQTQDIVFQM 6.719 181. 32 47 QLRRVFGNCL
6.169 182. 33 188 SLGSSEVENC 5.599 183. 34 329 LLDTDGPQRT 5.067
184. 35 25 SVHLDGFLFC 4.543 185. 36 39 SQLHGWNEQL 3.927 186. 37 288
AVLSVGNNEI 3.378 187. 38 311 IVFQMSRVKC 2.734 188. 39 319
KCWQVTFLGT 2.685 189. 40 198 KVGLRKWYMA 2.586 190. 41 228
AIQDIEKGWA 2.003 191. 42 190 GSSEVENCKV 1.861 192. 43 27 HLDGFLFCRV
1.797 193. 44 17 LGGRYVLYSV 1.775 194. 45 192 SEVENCKVGL 1.703 195.
46 323 VTFLGTLLDT 1.497 196. 47 385 AENTEMQIEV 1.352 197. 48 56
LPPFPPKYYL 1.304 198. 49 98 FVEFLKLAQL 1.266 199. 50 96 DVFVEFLKLA
1.054 200.
[0866]
17TABLE IX HLA Peptide Scoring Results - 158P1H4 - A3, 9-mers Start
Subsequence Residue Score (Estimate of Half Time of Disassociation
of a Rank Position Listing Molecule Containing This Subsequence)
Seq.ID# 1 161 GLFLIRFGK 1350.000 201. 2 374 KMISEKMVK 180.000 202.
3 27 HLDGFLFCR 81.000 203. 4 178 KLADFELPY 72.000 204. 5 366
FLLSSCLKK 60.000 205. 6 151 GLCRELLGY 36.000 206. 7 371 CLKKMISEK
30.000 207. 8 355 WQWFVIYTK 20.250 208. 9 141 RVLEVVSHK 20.250 209.
10 55 CLPPFPPKY 18.000 210. 11 416 FLSRKSKIK 10.000 211. 12 108
NTFDIATKK 7.500 212. 13 338 TLNQNLELR 6.000 213. 14 16 ALGGRYVLY
6.000 214. 15 273 QLDPCTCDY 6.000 215. 16 329 LLDTDGPQR 4.000 216.
17 259 FLELAREVR 4.000 217. 18 122 FLPNEQSIR 4.000 218. 19 155
ELLGYFGLF 3.645 219. 20 156 LLGYFGLFL 3.600 220. 21 391 QIEVPEQSK
3.000 221. 22 40 QLHGWNEQL 2.700 222. 23 54 NCLPPFPPK 2.025 223. 24
164 LIRFGKEGK 2.000 224. 25 244 RQKLEAFQK 1.800 225. 26 353
SCWQWFVIY 1.800 226. 27 226 MQAIQDIEK 1.200 227. 28 311 IVFQMSRVK
1.000 228. 29 250 FQKEDSQTK 0.900 229. 30 78 QLEQYLQNV 0.900 230.
31 142 VLEVVSHKI 0.900 231. 32 209 SLDSVLMDC 0.900 232. 33 190
GSSEVENCK 0.675 233. 34 25 SVHLDGFLF 0.600 234. 35 382 KLAAENTEM
0.600 235. 36 424 KIAKDDCVF 0.600 236. 37 289 VLSVGNNEI 0.600 237.
38 317 RVKCWQVTF 0.600 238. 39 106 QLNTFDIAT 0.600 239. 40 158
GYFGLFLIR 0.540 240. 41 96 DVFVEFLKL 0.540 241. 42 172 KLSVVKKLA
0.450 242. 43 174 SVVKKLADF 0.450 243. 44 393 EVPEQSKSK 0.450 244.
45 205 YMAPSLDSV 0.450 245. 46 86 VTMDPNVLR 0.450 246. 47 133
IITSDTAER 0.400 247. 48 256 QTKFLELAR 0.400 248. 49 411 KDYSSFLSR
0.360 249. 50 87 TMDPNVLRS 0.360 250.
[0867]
18TABLE X HLA Peptide Scoring Results - 158P1H4 - A3, 10-mers Start
Subsequence Residue Score (Estimate of Half Time of Disassociation
of a Rank Position Listing Molecule Containing This Subsequence)
Seq.ID# 1 225 YMQAIQDIEK 40.000 251. 2 163 FLIRFGKEGK 30.000 252. 3
106 QLNTFDIATK 30.000 253. 4 22 VLYSVHLDGF 15.000 254. 5 92
VLRSDVFVEF 9.000 255. 6 209 SLDSVLMDCR 6.000 256. 7 40 QLHGWNEQLR
6.000 257. 8 328 TLLDTDGPQR 6.000 258. 9 103 KLAQLNTFDI 5.400 259.
10 55 CLPPFPPKYY 4.500 260. 11 223 LLYMQAIQDI 4.500 261. 12 374
KMISEKMVKL 4.050 262. 13 338 TLNQNLELRF 4.000 263. 14 66 AMTTAMADER
4.000 264. 15 101 FLKLAQLNTF 3.000 265. 16 151 GLCRELLGYF 2.700
266. 17 156 LLGYFGLFLI 2.700 267. 18 155 ELLGYFGLFL 2.430 268. 19
50 RVFGNCLPPF 2.250 269. 20 390 MQIEVPEQSK 2.025 270. 21 47
QLRRVFGNCL 1.800 271. 22 229 IQDIEKGWAK 1.800 272. 23 411
KDYSSFLSRK 1.350 273. 24 145 VVSHKIGLCR 1.200 274. 25 416
FLSRKSKIKI 1.200 275. 26 122 FLPNEQSIRI 1.200 276. 27 16 ALGGRYVLYS
1.080 277. 28 364 QAFLLSSCLK 1.000 278. 29 214 LMDCRVAVDL 0.900
279. 30 393 EVPEQSKSKK 0.900 280. 31 205 YMAPSLDSVL 0.900 281. 32
178 KLADFELPYV 0.900 282. 33 359 VIYTKQAELL 0.900 283. 34 27
HLDGFLFCRV 0.900 284. 35 308 TQDIVFQMSR 0.720 285. 36 314
QMSRVKCWQV 0.600 286. 37 85 NVTMDPNVLR 0.600 287. 38 70 AMADERRDQL
0.600 288. 39 302 TLPDSQTQDI 0.600 289. 40 188 SLGSSEVENC 0.600
290. 41 169 KEGKLSVVKK 0.540 291. 42 53 GNCLPPFPPK 0.540 292. 43
200 GLRKWYMAPS 0.540 293. 44 94 RSDVFVEFLK 0.450 294. 45 113
ATKKAYLDIF 0.450 295. 46 379 KMVKLAAENT 0.450 296. 47 31 FLFCRVRYSQ
0.450 297. 48 370 SCLKKMISEK 0.450 298. 49 337 RTLNQNLELR 0.450
299. 50 158 GYFGLFLIRF 0.405 300.
[0868]
19TABLE XI HLA Peptide Scoring Results - 158P1H4 - A11, 9-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 141 RVLEVVSHK 9.000 301. 2 161 GLFLIRFGK
7.200 302. 3 244 RQKLEAFQK 5.400 303. 4 374 KMISEKMVK 3.600 304. 5
355 WQWFVIYTK 2.400 305. 6 108 NTFDIATKK 2.000 306. 7 366 FLLSSCLKK
1.200 307. 8 226 MQAIQDIEK 1.200 308. 9 158 GYFGLFLIR 0.960 309. 10
250 FQKEDSQTK 0.600 310. 11 86 VTMDPNVLR 0.400 311. 12 371
CLKKMISEK 0.400 312. 13 256 QTKFLELAR 0.400 313. 14 164 LIRFGKEGK
0.400 314. 15 391 QIEVPEQSK 0.400 315. 16 311 IVFQMSRVK 0.400 316.
17 393 EVPEQSKSK 0.300 317. 18 365 AFLLSSCLK 0.300 318. 19 54
NCLPPFPPK 0.300 319. 20 27 HLDGFLFCR 0.240 320. 21 67 MTTAMADER
0.200 321. 22 416 FLSRKSKIK 0.200 322. 23 68 TTAMADERR 0.200 323.
24 394 VPEQSKSKK 0.200 324. 25 169 KEGKLSVVK 0.180 325. 26 419
RKSKIKIAK 0.120 326. 27 198 KVGLRKWYM 0.120 327. 28 218 RVAVDLLYM
0.120 328. 29 412 DYSSFLSRK 0.120 329. 30 95 SDVFVEFLK 0.090 330.
31 337 RTLNQNLEL 0.090 331. 32 133 IITSDTAER 0.080 332. 33 338
TLNQNLELR 0.080 333. 34 259 FLELAREVR 0.080 334. 35 122 FLPNEQSIR
0.080 335. 36 329 LLDTDGPQR 0.080 336. 37 230 QDIEKGWAK 0.060 337.
38 238 KPTQAQRQK 0.060 338. 39 190 GSSEVENCK 0.060 339. 40 5
FCIPVSQQR 0.060 340. 41 317 RVKCWQVTF 0.060 341. 42 25 SVHLDGFLF
0.060 342. 43 411 KDYSSFLSR 0.048 343. 44 220 AVDLLYMQA 0.040 344.
45 236 WAKPTQAQR 0.040 345. 46 107 LNTFDIATK 0.040 346. 47 403
YHIQQSQQK 0.030 347. 48 358 FVIYTKQAF 0.030 348. 49 91 NVLRSDVFV
0.030 349. 50 174 SVVKKLADF 0.030 350.
[0869]
20TABLE XII HLA Peptide Scoring Results - 158P1H4 - A11, 10-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 229 IQDIEKGWAK 1.200 351. 2 402 KYHIQQSQQK
1.200 352. 3 337 RTLNQNLELR 0.900 353. 4 390 MQIEVPEQSK 0.900 354.
5 225 YMQAIQDIEK 0.800 355. 6 145 VVSHKIGLCR 0.800 356. 7 163
FLIRFGKEGK 0.600 357. 8 365 AFLLSSCLKK 0.600 358. 9 393 EVPEQSKSKK
0.600 359. 10 364 QAFLLSSCLK 0.400 360. 11 106 QLNTFDIATK 0.400
361. 12 85 NVTMDPNVLR 0.400 362. 13 370 SCLKKMISEK 0.300 363. 14
373 KKMISEKMVK 0.240 364. 15 308 TQDIVFQMSR 0.240 365. 16 255
SQTKFLELAR 0.240 366. 17 67 MTTAMADERR 0.200 367. 18 249 AFQKEDSQTK
0.200 368. 19 94 RSDVFVEFLK 0.180 369. 20 258 KFLELAREVR 0.180 370.
21 169 KEGKLSVVKK 0.180 371. 22 415 SFLSRKSKIK 0.150 372. 23 53
GNCLPPFPPK 0.120 373. 24 411 KDYSSFLSRK 0.120 374. 25 50 RVFGNCLPPF
0.120 375. 26 198 KVGLRKWYMA 0.120 376. 27 328 TLLDTDGPQR 0.120
377. 28 194 VENCKVGLRK 0.120 378. 29 193 EVENCKVGLR 0.120 379. 30
11 QQRSDALGGR 0.120 380. 31 160 FGLFLIRFGK 0.090 381. 32 141
RVLEVVSHKI 0.090 382. 33 66 AMTTAMADER 0.080 383. 34 209 SLDSVLMDCR
0.080 384. 35 40 QLHGWNEQLR 0.080 385. 36 168 GKEGKLSVVK 0.060 386.
37 243 QRQKLEAFQK 0.060 387. 38 427 KDDCVFGNIK 0.060 388. 39 317
RVKCWQVTFL 0.060 389. 40 35 RVRYSQLHGW 0.060 390. 41 121 IFLPNEQSIR
0.060 391. 42 175 VVKKLADFEL 0.060 392. 43 158 GYFGLFLIRF 0.048
393. 44 392 IEVPEQSKSK 0.045 394. 45 107 LNTFDIATKK 0.040 395. 46
418 SRKSKIKIAK 0.040 396. 47 4 HFCTPVSQQR 0.040 397. 48 354
CWQWFVIYTK 0.040 398. 49 103 KLAQLNTFDI 0.036 399. 50 132
EIITSDTAER 0.036 400.
[0870]
21TABLE XIII HLA Peptide Scoring Results - 158P1H4 - A24, 9-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 20 RYVLYSVHL 600.000 401. 2 360 IYTKQAFLL
200.000 402. 3 23 LYSVHLDGF 100.000 403. 4 224 LYMQAIQDI 90.000
404. 5 63 YYLAMTTAM 37.500 405. 6 181 DFELPYVSL 30.000 406. 7 117
AYLDIFLPN 15.120 407. 8 337 RTLNQNLEL 13.200 408. 9 37 RYSQLHGWN
12.000 409. 10 121 IFLPNEQSI 10.800 410. 11 159 YFGLFLIRF 10.000
411. 12 62 KYYLAMTTA 10.000 412. 13 51 VFGNCLPPF 10.000 413. 14 270
GYLQLDPCT 9.000 414. 15 280 DYPESGSGA 9.000 415. 16 415 SFLSRKSKI
8.250 416. 17 94 RSDVFVEFL 8.000 417. 18 149 KIGLCRELL 8.000 418.
19 254 DSQTKFLEL 7.920 419. 20 204 WYMAPSLDS 7.500 420. 21 24
YSVHLDGFL 7.200 421. 22 206 MAPSLDSVL 7.200 422. 23 144 EVVSHKIGL
6.000 423. 24 15 DALGGRYVL 6.000 424. 25 9 VSQQRSDAL 6.000 425. 26
193 EVENCKVGL 6.000 426. 27 321 WQVTFLGTL 6.000 427. 28 432
FGNIKEEDL 6.000 428. 29 96 DVFVEFLKL 5.280 429. 30 375 MISEKMVKL
5.280 430. 31 85 NVTMDPNVL 4.800 431. 32 71 MADERRDQL 4.800 432. 33
364 QAFLLSSCL 4.800 433. 34 322 QVTFLGTLL 4.800 434. 35 409
QQKDYSSFL 4.800 435. 36 135 TSDTAERVL 4.000 436. 37 317 RVKCWQVTF
4.000 437. 38 40 QLHGWNEQL 4.000 438. 39 331 DTDGPQRTL 4.000 439.
40 216 DCRVAVDLL 4.000 440. 41 111 DIATKKAYL 4.000 441. 42 33
FCRVRYSQL 4.000 442. 43 156 LLGYFGLFL 4.000 443. 44 359 VIYTKQAFL
4.000 444. 45 424 KIAKDDCVF 4.000 445. 46 44 WNEQLRRVF 3.600 446.
47 155 ELLGYFGLF 3.600 447. 48 339 LNQNLELRF 3.600 448. 49 358
FVIYTKQAF 3.600 449. 50 305 DSQTQDIVF 3.000 450.
[0871]
22TABLE XIV HLA Peptide Scoring Results - 158P1H4 - A24, 10-mers
Subsequence Score (Estimate of Half Time Start Residue of
Disassociation of a Molecule Rank Position Listing Containing This
Subsequence) Seq.ID# 1 23 LYSVHLDGFL 240.000 451. 2 158 GYFGLFLIRF
100.000 452. 3 62 KYYLAMTTAM 50.000 453. 4 431 VFGNIKEEDL 20.000
454. 5 32 LFCRVRYSQL 20.000 455. 6 357 WFVIYTKQAF 18.000 456. 7 374
KMISEKMVKL 13.200 457. 8 238 KPTQAQRQKL 10.560 458. 9 363
KQAFLLSSCL 9.600 459. 10 280 DYPESGSGAV 9.000 460. 11 334
GPQRTLNQNL 8.640 461. 12 317 RVKCWQVTFL 8.000 462. 13 204
WYMAPSLDSV 7.500 463. 14 270 GYLQLDPCTC 7.500 464. 15 63 YYLAMTTAMA
7.500 465. 16 84 QNVTMDPNVL 7.200 466. 17 321 WQVTFLGTLL 7.200 467.
18 412 DYSSFLSRKS 6.600 468. 19 170 EGKLSVVKKL 6.160 469. 20 98
FVEFLKLAQL 6.000 470. 21 39 SQLHGWNEQL 6.000 471. 22 348 QYSEDSCWQW
6.000 472. 23 155 ELLGYFGLFL 6.000 473. 24 358 FVIYTKQAFL 6.000
474. 25 360 IYTKQAFLLS 6.000 475. 26 320 CWQVTFLGTL 6.000 476. 27
56 LPPFPPKYYL 6.000 477. 28 294 NNEISCCITL 6.000 478. 29 408
SQQKDYSSFL 6.000 479. 30 281 YPESGSGAVL 6.000 480. 31 205
YMAPSLDSVL 5.760 481. 32 214 LMDCRVAVDL 5.600 482. 33 141
RVLEVVSHKI 5.544 483. 34 268 HYGYQLDPC 5.000 484. 35 70 AMADERRDQL
4.800 485. 36 134 ITSDTAERVL 4.800 486. 37 47 QLRRVFGNCL 4.800 487.
38 175 VVKKLADFEL 4.400 488. 39 164 LIRFGKEGKL 4.400 489. 40 349
YSEDSCWQWF 4.320 490. 41 43 GWNEQLRRVF 4.320 491. 42 50 RVFGNCLPPF
4.000 492. 43 359 VIYTKQAFLL 4.000 493. 44 265 EVRHYGYLQL 4.000
494. 45 92 VLRSDVFVEF 3.696 495. 46 338 TLNQNLELRF 3.600 496. 47 89
DPNVLRSDVF 3.000 497. 48 407 QSQQKDYSSF 3.000 498. 49 24 YSVHLDGFLF
3.000 499. 50 241 QAQRQKLEAF 3.000 500.
[0872]
23TABLE XV HLA Peptide Scoring Results - 158P1H4 - B7, 9-mers Start
Subsequence Residue Score (Estimate of Half Time of Disassociation
of a Rank Position Listing Molecule Containing This Subsequence)
Seq.ID# 1 207 APSLDSVLM 60.000 501. 2 216 DCRVAVDLL 40.000 502. 3
33 FCRVRYSQL 40.000 503. 4 96 DVFVEFLKL 20.000 504. 5 59 FPPKYYLAM
20.000 505. 6 144 EVVSHKJGL 20.000 506. 7 85 NVTMDPNVL 20.000 507.
8 322 QVTFLGTLL 20.000 508. 9 15 DALGGRYVL 18.000 509. 10 364
QAFLLSSCL 12.000 510. 11 57 PPFPPKYYL 12.000 511. 12 206 MAPSLDSVL
12.000 512. 13 123 LPNEQSIRI 8.000 513. 14 193 EVENCKVGL 6.000 514.
15 89 DPNYLRSDV 6.000 515. 16 149 KIGLCRELL 6.000 516. 17 71
MADERRDQL 5.400 517. 18 218 RVAVDLLYM 5.000 518. 19 198 KVGLRKWYM
5.000 519. 20 337 RTLNQNLEL 4.000 520. 21 375 MISEKMVKL 4.000 521.
22 24 YSVHLDGFL 4.000 522. 23 48 LRRVFGNCL 4.000 523. 24 111
DIATKKAYL 4.000 524. 25 321 WQVTFLGTL 4.000 525. 26 335 PQRTLNQNL
4.000 526. 27 417 LSRKSKIKI 4.000 527. 28 9 VSQQRSDAL 4.000 528. 29
254 DSQTKFLEL 4.000 529. 30 409 QQKDYSSFL 4.000 530. 31 359
VIYTKQAFL 4.000 531. 32 156 LLGYFGLFL 4.000 532. 33 432 FGNIKEEDL
4.000 533. 34 40 QLHGWNEQL 4.000 534. 35 303 LPDSQTQDI 2.400 535.
36 315 MSRVKCWQV 2.000 536. 37 18 GGRYVLYSV 2.000 537. 38 331
DTDGPQRTL 1.800 538. 39 94 RSDVFVEFL 1.200 539. 40 113 ATKKAYLDI
1.200 540. 41 281 YPESGSGAV 1.200 541. 42 104 LAQLNTFDI 1.200 542.
43 135 TSDTAERVL 1.200 543. 44 384 AAENTEMQI 1.080 544. 45 307
QTQDIVFQM 1.000 545. 46 382 KLAAENTEM 1.000 546. 47 186 YVSLGSSEV
1.000 547. 48 91 NVLRSDVFV 1.000 548. 49 47 QLRRVFGNC 1.000 549. 50
367 LLSSCLKKM 1.000 550.
[0873]
24TABLE XVI HLA Peptide Scoring Results - 158P1H4 - B7, 10-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 265 EVRHYGYLQL 200.000 551. 2 56 LPPFPPKYYL
120.000 552. 3 238 KPTQAQRQKL 120.000 553. 4 334 GPQRTLNQNL 80.000
554. 5 47 QLRRVFGNCL 40.000 555. 6 164 LIRFGKEGKL 40.000 556. 7 281
YPESGSGAVL 24.000 557. 8 358 FVIYTKQAFL 20.000 558. 9 175
VVKKLADFEL 20.000 559. 10 317 RVKCWQVTFL 20.000 560. 11 70
AMADERRDQL 18.000 561. 12 98 FVEFLKLAQL 6.000 562. 13 288
AVLSVGNNEI 6.000 563. 14 408 SQQKDYSSFL 4.000 564. 15 39 SQLHGWNEQL
4.000 565. 16 84 QNVTMDPNVL 4.000 566. 17 170 EGKLSVVKKL 4.000 567.
18 134 ITSDTAERVL 4.000 568. 19 321 WQVTFLGTLL 4.000 569. 20 74
ERRDQLEQYL 4.000 570. 21 374 KMISEKMVKL 4.000 571. 22 205
YMAPSLDSVL 4.000 572. 23 155 ELLGYFGLFL 4.000 573. 24 359
VIYTKQAFLL 4.000 574. 25 363 KQAFLLSSCL 4.000 575. 26 206
MAPSLDSVLM 3.000 576. 27 7 IPVSQQRSDA 3.000 577. 28 141 RVLEVVSHKI
2.000 578. 29 8 PVSQQRSDAL 2.000 579. 30 59 FPPKYYLAMT 2.000 580.
31 180 ADFELPYVSL 1.800 581. 32 220 AVDLLYMQAI 1.800 582. 33 212
SVLMDCRVAV 1.500 583. 34 112 IATKKAYLDI 1.200 584. 35 383
LAAENTEMQI 1.200 585. 36 294 NNEISCCITL 1.200 586. 37 303
LPDSQTQDIV 1.200 587. 38 214 LMDCRVAVDL 1.200 588. 39 315
MSRVKCWQVT 1.000 589. 40 306 SQTQDIVFQM 1.000 590. 41 366
FLLSSCLKKM 1.000 591. 42 371 CLKKMISEKM 1.000 592. 43 417
LSRKSKIKIA 1.000 593. 44 35 RVRYSQLHGW 1.000 594. 45 311 IVFQMSRVKC
0.750 595. 46 262 LAREVRHYGY 0.600 596. 47 207 APSLDSVLMD 0.600
597. 48 330 LDTDGPQRTL 0.600 598. 49 14 SDALGGRYVL 0.600 599. 50
148 HKIGLCRELL 0.600 600.
[0874]
25TABLE XVII HLA Peptide Scoring Results - 158P1H4 - B35, 9-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 207 APSLDSVLM 60.000 601. 2 56 LPPFPPKYY
40.000 602. 3 59 FPPKYYLAM 40.000 603. 4 123 LPNEQSIRI 16.000 604.
5 178 KLADFELPY 8.000 605. 6 218 RVAVDLLYM 6.000 606. 7 397
QSKSKKYHI 6.000 607. 8 409 QQKDYSSFL 6.000 608. 9 382 KLAAENTEM
6.000 609. 10 417 LSRKSKIKI 6.000 610. 11 13 RSDALGGRY 6.000 611.
12 317 RVKCWQVTF 6.000 612. 13 152 LCRELLGYF 6.000 613. 14 305
DSQTQDIVF 5.000 614. 15 9 VSQQRSDAL 5.000 615. 16 254 DSQTKFLEL
5.000 616. 17 24 YSVHLDGFL 5.000 617. 18 341 QNLELRFQY 4.000 618.
19 198 KVGLRKWYM 4.000 619. 20 89 DPNVLRSDV 4.000 620. 21 307
QTQDIVFQM 4.000 621. 22 15 DALGGRYVL 3.000 622. 23 151 GLCRELLGY
3.000 623. 24 33 FCRVRYSQL 3.000 624. 25 242 AQRQKLEAF 3.000 625.
26 424 KIAKDDCVF 3.000 626. 27 315 MSRVKCWQV 3.000 627. 28 206
MAPSLDSVL 3.000 628. 29 216 DCRVAVDLL 3.000 629. 30 364 QAFLLSSCL
3.000 630. 31 261 ELAREVRHY 3.000 631. 32 94 RSDVFVEFL 3.000 632.
33 303 LPDSQTQDI 2.400 633. 34 352 DSCWQWFVI 2.000 634. 35 375
MISEKMVKL 2.000 635. 36 57 PPFPPKYYL 2.000 636. 37 80 EQYLQNVTM
2.000 637. 38 353 SCWQWFVIY 2.000 638. 39 184 LPYVSLGSS 2.000 639.
40 149 KIGLCRELL 2.000 640. 41 334 GPQRTLNQN 2.000 641. 42 367
LLSSCLKKM 2.000 642. 43 368 LSSCLKKMI 2.000 643. 44 405 IQQSQQKDY
2.000 644. 45 337 RTLNQNLEL 2.000 645. 46 55 CLPPFPPKY 2.000 646.
47 16 ALGGRYVLY 2.000 647. 48 71 MADERRDQL 1.800 648. 49 349
YSEDSCWQW 1.500 649. 50 85 NVTMDPNVL 1.500 650.
[0875]
26TABLE XVIII HLA Peptide Scoring Results - 158P1H4 - B35, 10-mers
Start Subsequence Residue Score (Estimate of Half Time of
Disassociation of a Rank Position Listing Molecule Containing This
Subsequence) Seq.ID# 1 238 KPTQAQRQKL 40.000 651. 2 262 LAREVRHYGY
36.000 652. 3 334 GPQRTLNQNL 20.000 653. 4 56 LPPFPPKYYL 20.000
654. 5 89 DPNVLRSDVF 20.000 655. 6 394 VPEQSKSKKY 12.000 656. 7 352
DSCWQWFVIY 10.000 657. 8 206 MAPSLDSVLM 9.000 658. 9 24 YSVHLDGFLF
7.500 659. 10 407 QSQQKDYSSF 7.500 660. 11 250 FQKEDSQTKF 6.000
661. 12 196 NCKVGLRKWY 6.000 662. 13 317 RVKCWQVTFL 6.000 663. 14
281 YPESGSGAVL 6.000 664. 15 216 DCRVAVDLLY 6.000 665. 16 15
DALGGRYVLY 6.000 666. 17 371 CLKKMISEKM 6.000 667. 18 173
LSVVKKLADF 5.000 668. 19 272 LQLDPCTCDY 4.000 669. 20 150
IGLCRELLGY 3.000 670. 21 35 RVRYSQLHGW 3.000 671. 22 265 EVRHYGYLQL
3.000 672. 23 275 DPCTCDYPES 3.000 673. 24 175 VVKKLADFEL 3.000
674. 25 101 FLKLAQLNTF 3.000 675. 26 399 KSKKYHIQQS 3.000 676. 27
164 LIRFGKEGKL 3.000 677. 28 92 VLRSDVFVEF 3.000 678. 29 47
QLRRVFGNCL 3.000 679. 30 113 ATKKAYLDIF 3.000 680. 31 170
EGKLSVVKKL 3.000 681. 32 349 YSEDSCWQWF 3.000 682. 33 190
GSSEVENCKV 3.000 683. 34 241 QAQRQKLEAF 3.000 684. 35 383
LAAENTEMQI 2.400 685. 36 70 AMADERRDQL 2.000 686. 37 7 IPVSQQRSDA
2.000 687. 38 366 FLLSSCLKKM 2.000 688. 39 306 SQTQDIVFQM 2.000
689. 40 50 RVFGNCLPPF 2.000 690. 41 363 KQAFLLSSCL 2.000 691. 42
414 SSFLSRKSKI 2.000 692. 43 29 DGFLFCRVRY 2.000 693. 44 340
NQNLELRFQY 2.000 694. 45 55 CLPPFPPKYY 2.000 695. 46 374 KMISEKMVKL
2.000 696. 47 404 HIQQSQQKDY 2.000 697. 48 59 FPPKYYLAMT 2.000 698.
49 54 NCLPPFPPKY 2.000 699. 50 134 ITSDTAERVL 2.000 700.
[0876]
27TABLE XIX Motif-bearing Subsequences of the 158P1H4 Protein
Protein Motifs of 158P1H4 N-glycosylation site 85-88 NVTM Protein
kinase C phosphorylation site 1 114-116 TKK 2 128-130 SIR 3 147-149
SHK 4 377-379 SEK 5 400-402 SKK 6 418-420 SRK Casein kinase II
phosphorylation site 1 192-195 SEVE 2 302-305 TLPD 3 328-331 TLLD
Tyrosine kinase phosphorylation site 218-225 RVAVDLLY
N-myristoylation site 287-292 GAVLSV One PX domain was identified
by Pfam 52-102 FGNCLPPFPPKYYLAMTTAMADERRDQLEQYLQNVTMDPNVLRS-
DVFVEFL (SEQ ID NO:736)
[0877]
28TABLE XX Frequently Occurring Motifs avrg. % Name identity
Description Potential Function zf-C2H2 34% zinc finger, Nucleic
acid-binding C2H2 type protein functions as transcription factor,
nuclear location probable cytochrome_b_N 68% Cytochrome membrane
bound b(N-terminal)/ oxidase, generate b6/petB superoxide ig 19%
Immuno- domains are one hundred globulin amino acids long and
domain include a conserved intradomain disulfide bond. WD40 18% WD
domain, tandem repeats of about G-beta repeat 40 residues, each
containing a Trp--Asp motif. Function in signal transduction and
protein interaction PDZ 23% PDZ domain may function in targeting
signaling molecules to sub-membranous sites LRR 28% Leucine Rich
short sequence motifs Repeat involved in protein-- protein
interactions pkinase 23% Protein kinase conserved catalytic core
domain common to both serine/ threonine and tyrosine protein
kinases con- taining an ATP binding site and a catalytic site PH
16% PH domain pleckstrin homology involved intracellular signaling
or as constituents of the cytoskeleton EGF 34% EGF-like 30-40
amino-acid long domain found in the extracellular domain of
membrane- bound proteins or in secreted proteins rvt 49% Reverse
transcriptase RNA- dependent DNA polymerase) ank 25% Ank repeat
Cytoplasmic protein, associates integral membrane proteins to the
cytoskeleton oxidored_q1 32% NADH- membrane associated. Ubiquinone/
Involved in proton plastoquinone translocation across the (complex
I), membrane various chains efhand 24% EF hand calcium-binding
domain, consists of a12 residue loop flanked on both sides by a 12
residue alpha-helical domain rvp 79% Retroviral Aspartyl or acid
aspartyl proteases, centered on a protease catalytic aspartyl
residue Collagen 42% Collagen triple extracellular structural helix
repeat proteins involved in 20 copies) formation of connective
tissue. The sequence consists of the G--X--Y and the polypeptide
chains forms a triple helix. fn3 20% Fibronectin Located in the
extra- type III domain cellular ligand-binding region of receptors
and is about 200 amino acid residues long with two pairs of
cysteines involved in disulfide bonds 7tm_1 19% 7 trans- seven
hydrophobic trans- membrane membrane regions, with receptor the
N-terminus located (rhodopsin extracellularly while the family)
C-terminus is cyto- plasmic. Signal through G proteins
[0878]
29TABLE XXI TNM CLASSIFICATION OF BLADDER TUMORS Primary tumor (T)
The suffix (m) should be added to the appropriate T category to
indicate multiple tumors. The suffix (is) may be added to any T to
indicate the presence of associated carcinoma in situ. TX Primary
tumor cannot be assessed TO No evidence of primary tumor Ta
Noninvasive papillary carcinoma Tis Carcinoma in situ: "flat tumor"
T1 Tumor invades sub-epithelial connective tissue T2 Tumor invades
superficial muscle (inner half) T3 Tumor invades deep muscle or
perivesical fat T3a Tumor invades deep muscle (outer half) T3b
Tumor invades perivesical fat i. microscopically ii.
macroscopically (extravesical mass) T4 Tumor invades any of the
following: prostate, uterus, vagina, pelvic wall, or abdominal wall
T4a Tumor invades the prostate, uterus, vagina T4b Tumor invades
the pelvic wall or abdominal wall or both Regional lymph nodes (N)
Regional lymph nodes are those within the true pelvis: all others
are distant nodes NX Regional lymph nodes cannot be assessed N0 No
regional lymph node metastasis N1 Metastasis in a single lymph
node, 2 cm or less in greatest dimension N2 Metastasis in a single
lymph node, more than 2 cm but not more than 5 cm in greatest
dimension, or multiple lymph nodes, none more than 5 cm in greatest
dimension N3 Metastasis in a lymph node more than 5 cm in greatest
dimension Distant metastasis (M) MX Presence of distant metastasis
cannot be assessed M0 No distant metastasis M1 Distant metastasis
Stage grouping Stage 0.sub.a Ta N0 M0 0.sub.is Tis N0 M0 I T1 N0 M0
II T2 N0 M0 T3a N0 M0 III T3b N0 M0 T4a N0 M0 IV T4b N0 M0 Any T
N1-3 M0 Any T Any N M1
[0879]
30TABLE XXIA Nucleotide sequence of the splice variant predicted by
FgenesH for 158P1H4. 1 atggacccaa acgtgttgag aagtgatgtc ttcgttgagt
ttttaaaact ggcgcagctg 61 aatacatttg acatcgccac caagaaagct
tatctggaca tatttctgcc caatgaacag 121 agtattagaa tcgaaattat
aacatcagac actgctgaaa gagtactaga gcatacattt 181 gatgaagtac
atttgcagtc agaaaaatgt gcttcctctc ttccaatcat gttggcttgt 241
gagaaattca gacccaagac gttgacatca tcagatcagg tggtgtcaca caaaattgga
301 ctgtgtcgag agctcttggg ctacttcggc ctctttctca ttcggtttgg
caaggagggc 361 aagctctctg tgaaaaaatt ggctgacttt gaactccctt
atgttagtct tggaagttct 421 gaggtggaaa actgtaaggt tggactccga
aagtatatgg ctccatccct cgactccgtg 481 ctgatggact gcagggtggc
ggtagatttg ctctacatgc aggcaataca ggacattgaa 541 aaaggatggg
ccaaacccac acaggcacag aggcagaaat tagaagcttt ccagaaagaa 601
gacagtcaaa caaagttttt ggagctggcc cgggaggtac ggcactatgg atacctgcag
661 ctggatcctt gtacctgtga ctacccagaa tcaggctctg gagctgttct
ttctgttggc 721 aataatgaga tcagctgctg catcaccctg cctgacagcc
agacccagga catcgttttc 781 cagatgagca gggtgaagtg ctggcaggtc
actttccttg tgagtatctt ggcaccttgc 841 tttgcccatg acttaaaaat
cctagtaccc ttggatgaca aagttgacac tagggagaca 901 ggtttgccat
ttgaatctgt accttga
[0880]
31TABLE XXIIA Nucleotide sequence alignment of the splice variant
predicted by EgenesH with 158P1H4. Score = 1002 bits (521), Expect
= 0.0 Identities = 543/549 (98%), Gaps = 6/549 (1%) Strand =
Plus/Plus 158P1H4: 494
aggtggtgtcacacaaaattggactgtgtcgagagctcttgggctacttcggcctctttc 553
.vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline. FgenesH: 278
aggtggtgtcacacaaaattggactgtgtc- gagagctcttgggctacttcggcctctttc 337
158P1H4: 554
tcattcggtttggcaaggagggcaagctctctgttgtgaaaaaattggctgactttgaac 613
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline.
.vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline. FgenesH:
338 tcattcggtttggcaaggagggcaagctctctgt---gaaaaaattggctgactttgaac
394 158P1H4: 614 tcccttatgttagtcttggaagttctgaggtggaaaactgtaaggttgg-
actccgaaagt 673 .vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline. FgenesH: 395
tcccttatgttagtcttggaagttctgaggtggaaaactgtaaggttggactccgaaagt 454
158P1H4: 674 ggtatatggctccatccctcgactccgtgctgatggactgcagggtggcggta-
gatttgc 733 .vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e. FgenesH: 455
---atatggctccatccctcgactccgtgctgatggactgcagggtggcgg- tagatttgc 511
158P1H4: 734 tctacatgcaggcaatacaggacattgaaaa-
aggatgggccaaacccacacaggcacaga 793 .vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline. FgenesH: 512
tctacatgcaggcaatacaggacattgaaaaaggatgggccaaacccacacaggcacaga 571
158P1H4: 794 ggcagaaattagaagctttccagaaagaagacagtcaaacaaagtttttggag-
ctggccc 853 .vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline. FgenesH: 572
ggcagaaattagaagctttccaga- aagaagacagtcaaacaaagtttttggagctggccc 631
158P1H4: 854
gggaggtacggcactatggatacctgcagctggatccttgtacctgtgactacccagaat 913
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline. FgenesH: 632
gggaggtacggcactatggatacctgcagctggatccttgtacc- tgtgactacccagaat 691
158P1H4: 914 caggctctggagctgttctttctg-
ttggcaataatgagatcagctgctgcatcaccctgc 973 .vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline. FgenesH:
692 caggctctggagctgttctttctgttggcaataatgagatcagctgctgcatcaccctgc
751 158P1H4: 974 ctgacagccagacccaggacatcgttttccagatgagcagggtgaagtg-
ctggcaggtca 1033 .vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline. FgenesH: 752
ctgacagccagacccaggacatcgttttccagatgagcagggtgaagtgctggcaggtca 811
158P1H4: 1034 ctttccttg 1042 .vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline.
FgenesH: 812 ctttccttg 820 Score = 329 bits (171), Expect = 6e-87
Identities = 171/171 (100%) Strand = Plus/Plus 158P1H4: 325
atggacccaaacgtgttgagaagtgatgtcttcgttgagtttttaaaa- ctggcgcagctg 384
.vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline. FgenesH: 1
atggacccaaacgtgttgagaagtgatgtcttcgttgagtttttaaaactggcgcagctg 60
158P1H4: 385 aataeatttgacatcgccaccaagaaagcttatctggacatatttctgcccaa-
tgaacag 444 .vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline. FgenesH: 61
aatacatttgacatcgccaccaaga- aagcttatctggacatatttctgcccaatgaacag 120
158P1H4: 445 agtattagaatcgaaattataacatcagacactgctgaaagagtcctagag
495
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline.
FgenesH: 121 agtattagaatcgaaattataacatcagacactgctgaaagagtcctagag
171
[0881]
32TABLE XXIIIA Amino acid sequence alignment of the splice variant
for 158P1H4 predicted by FgenesH. Score = 132. bits (280), Expect =
4e-28 Identities = 59/68 (86%) Frame = +1/+1 158P1H4: 325
MDPNVLRSDVFVEFLKLAQLNTFDIATKKAYLDIFLPNEQSIRIEIITSDTAERVLEVVS 504
MDPNVLRSDVFVEFLKLAQLNTFDIATKKAYLDIFLPNEQSIRIEIITSDTAERVLE FgenesH:
1 MDPNVLRSDVFVEFLKLAQLNTFDIATKKAYLDIFLPNEQSIRIEIITSDTAERVLEHTF 180
Score = 78.0 bits (164), Expect(3) = e-110 Identities = 32/34 (94%)
Frame = +1/+1 158P1H4: 493 EVVSHKIGLCRELLGYFGLFLIRFGKEGKLSVVK 594
+VVSHKIGLCRELLGYFGLFLIRFGKEGKLSV K FgenesH: 277
QVVSHKIGLCRELLGYFGLFLIRFGKEGKLSVKK 378 Score = 68.4 bits (143),
Expect(3) = e-12.0 Identities = 28/29 (96%) Frame = +1/+1 158P1H4:
589 VKKLADFELPYVSLGSSEVENCKVGLRKW 675 VKKLADFELPYVSLGSSEVENCKVGLRK+
FgenesH: 370 VKKLADFELPYVSLGSSEVENCKVGLRKY 456 Score = 300 bits
(650), Expect(3) = e-110 Identities = 123/126 (97%) Frame = +1/+1
158P1H4: 676 YMAPSLDSVLMDCRVAVDLLYMQAIQDIEKGWAKPTQAQRQKLE-
AFQKEDSQTKFLELAR 855 YMAPSLDSVLMDCRVAVDLLYMQAIQDIEKGWAKPTQAQRQKL-
EAFQKEDSQTKFLELAR FgenesH: 454
YMAPSLDSVLMDCRVAVDLLYMQAIQDIEKGWAKPT- QAQRQKLEAFQKEDSQTKFLELAR 633
158P1H4: 856
EVRHYGYLQLDPCTCDYPESGSGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVT 1035
EVRHYGYLQLDPCTCDYPESGSGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVT
FgenesH: 634
EVRHYGYLQLDPCTCDYPESGSGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVT 813
158P1H4: 1036 FLGTLL 1053 FL ++L FgenesH: 814 FLVSIL 831
[0882]
33TABLE XXIVA Peptide sequences from the translation of the
nucleotide sequence of the splice variant predicted FgenesH. Open
reading frame Amino acid sequences Frame 1
MDPNVLRSDVFVEFLKLAQLNTFDIATKKAYLDIFLPNEQ- SIRIEIITSDTAERVLEHTF
DEVHLQSEKCASSLPIMLACEKFRPKTLTSSDQVVSHKIGLCRE- LLGYFGLFLIRFGKEG
KLSVKKLADFELPYVSLGSSEVENCKVGLRKYMAPSLDSVLMDCRVAVD- LLYMQAIQDIE
KGWAKPTQAQRQKLEAFQKEDSQTKFLELAREVRHYGYLQLDPCTCDYPESGSG- AVLSVG
NNEISCCITLPDSQTQDIVFQMSRVKCWQVTFLVSILAPCFAHDLKILVPLDDKVDTRE- T
GLPFESVP* Frame 2 WTQTC*EVMSSLSF*NWRS*IHLTSPPRKL-
IWTYFCPMNRVLESKL*HQTLLKEs*SIHL MKYICSQKNVLPLFQSCWLVRNSDPRR*HHQIRWC-
HTKLDCVESSWATSASFSFGLARRA SSL*KNWLTLNSLMLVLEVLRWKTVRLDSESIWLHPSTPC-
*WTAGWR*ICSTCRQYRTLK KDGPNPHRHRGRN*KLSRKKTVKQSFWSWPGRYGTMDTCSWILVP-
VTTQNQALELFFLLA IMRSAAASPCLTARPRTSFSR*AG*SAGRSLSL*VSWHLALPMT*KS*YP-
WMTKLTLGRQ VCHLNLYL Frame 3
GPKRVEK*CLR*VFKTGAAEYI*HRHQESLSGHISAQ*TEY*NRNYNIRHC*KSPRAYI*
*STFAVRKMCFLSSNHVGL*EIQTQDVDIIRSGGVTQNWTVSRALGLLRPLSHSVWQGGQ
ALCEKIG*L*TPLC*SWKF*GGKL*GWTPKVYGSIPRLRADGLQGGGRFALHAGNTGH*K
RMGQTHTGTEAEIRSFPERRQSNKVFGAGPGGTALWIPAAGSLYL*LPRIRLWSCSFCWQ
**DQLLHHPA*QPDPGHRFPDEQGEVLAGHFPCEYLGTLLCP*LKNPSTLG*QS*H*GDR
FAI*ICTL Note: Frame 1 gives the longest subsequence that is
identical with 158P1H4 amino acid sequence. In this TABLE each
(*)indicates the product of a single codon, i.e., a single unknown
amino acid or a stop codon.
[0883]
34TABLE XXIB Nucleotide sequence of the splice variant predicted by
GenScan for 158P1H4. 1 atggacccaa acgtgttgag aagtgatgtc ttcgttgagt
ttttaaaact ggcgcagctg 61 gttcaatttt caacgcactt ggcagccctt
ctgaaaacct caggctcagg gctatttagc 121 ttccctccat ttgcttcaag
attagcagcc ccagctgaag gcgaggaccc tgcccttcct 181 tccttcttcc
gagggagtga gaattgcact gagcatcgaa acctggagga cgtgccagac 241
aggaggagga aaggaaccca aaacatgggg gaaagagaga gacagaaaga aggggaaggg
301 aattgctttg aaagtggtga aactattcct cagctgctct ttattttcaa
agctatgctg 361 agaacaccca ctcataccag ccacctaatc agactgcttc
ctgggggtaa tttatgtatc 421 tctgacaaga gaaggccccc tgtgagcctg
gtatttgtac aactgccacc tagagaactg 481 ggagtcgtgg gtaaagcagc
tctggagggc agtgcttgga ggcctctcct tgcgtggtac 541 agaggtgatt
tagtggtgtc acacaaaatt ggactgtgtc gagagctctt gggctacttc 601
ggcctctttc tcattcggtt tggcaaggag ggcaagctct ctgttgtgaa aaaattggct
661 gactttgaac tcccttatgt tagtcttgga agttctgagg tggaaaactg
taaggttgga 721 ctccgaaagt gcttcggcca agtcatggaa tggtcagccg
gtgctgccgc caacatacac 781 acccaggtca ccagctgccc caggtgttca
cactcaccac accgccatct tcggagacct 841 gatcttttcc aggcttggct
ccacaattct gggaagaaag ctcagaagct tctttcagca 901 atacaggaca
ttgaaaaagg atgggccaaa cccacacagg cacagaggca gaaattagaa 961
gctttccaga aagaagacag tcaaacaaag tttttggagc tggcccggga ggtacggcac
1021 tatggatacc tgcagctgga tccttgtacc tgtgactacc cagaatcagg
ctctggagct 1081 gttctttctg ttggcaataa tgagatcagc tgctgcatca
ccctgcctga cagccagacc 1141 caggacatcg ttttccagat gagcagggtg
aagtgctggc aggtcacttt ccttgtgagt 1201 atcttggcac cttgctttgc
ccatgactta aaaatcctag tacccttgga tgacaaagtt 1261 gacactaggg
agacaggttt gccatttgaa tctgtacctt ga
[0884]
35TABLE XXIIB Nucleotide sequence alignment of the splice variant
predicted by GenScan with 158P1H4. Score = 573 bits (298), Expect =
e-160 Identities = 298/298 (100%) Strand = Plus/Plus 158P1H4: 745
gcaatacaggacattgaaaaaggatgggccaaacccacacaggcacagaggcagaaatta 804
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline. GenScan: 898
gcaatacaggacattgaaaaaggatgggccaaacccacacagg- cacagaggcagaaatta 957
158P1H4: 805 gaagctttccagaaagaagacag-
tcaaacaaagtttttggagctggcccgggaggtacgg 864 .vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline.
GenScan: 938
gaagctttccagaaagaagacagtcaaacaaagtttttggagctggcccgggaggtacgg 1017
158P1H4: 865 cactatggatacctgcagctggatccttgtacctgtgac-
tacccagaatcaggctctgga 924 .vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline. GenScan: 1018
cactatggatacctgcagctggatccttgtacctgtgactacccagaatcaggctctgga 1077
158P1H4: 925 gctgttctttctgttggcaataatgagatcagctgctgcatcaccctgcctg-
acagccag 984 .vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline..vertline..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline..vertline..vertline..vertline.-
.vertline..vertline..vertline. GenScan: 1078
gctgttctttctgttggcaataatgagatcagctgctgcatcaccctgcctgacagccag 1137
158P1H4: 985 acccaggacatcgttttccagatgagcagggtgaagtgctggcaggtcactt-
tccttg 1042 .vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline. GenScan: 1138
acccaggacatcgttttccagatgagcagggtgaagtgctggc- aggtcactttccttg 1195
Score = 344 bits (179), Expect = 2e-91 Identities = 179/179 (100%)
Strand = Plus/Plus 158P1H4: 496
gtggtgtcacacaaaattggactgtgtcgagagctcttgggctacttcgg- cctctttctc 555
.vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline. GenScan: 553
gtggtgtcacacaaaattggactgtgtcgagagctcttgggctacttcggcctctttctc 612
158P1H4: 556 attcggtttggcaaggagggcaagctctctgttgtgaaaaaattggctgactt-
tgaactc 615 .vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline. GenScan: 613
attcggtttggcaaggagggcaag- ctctctgttgtgaaaaaattggctgactttgaactc 672
158P1H4: 616
ccttatgttagtcttggaagttctgaggtggaaaactgtaaggttggactccgaaagtg 674
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline.
GenScan: 673 ccttatgttagtcttggaagttctgaggtggaaaactgtaaggttggactccg-
aaagtg 731 Score = 116 bits (60), Expect = 1e-22 Identities = 60/60
(100%) Strand = Plus/Plus 158P1H4: 325
atggacccaaacgtgttgagaagtgatgtcttcgttgagtttttaaaactggcgcagctg 384
.vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline. GenScan: 1
atggacccaaacgtgttgagaagtgatgtctt- cgttgagtttttaaaactggcgcagctg
60
[0885]
36TABLE XXIIIB Amino acid sequence alignment of the splice variant
for 158P1H4 predicted by GenScan. Score = 48.7 bits (100), Expect =
0.002 Identities = 21/23 (91%) Frame = +1/+1 158P1H4: 325
MDPNVLRSDVFVEFLKLAQLNTF 393 MDPNVLRSDVFVEFLKLAQL F GenScan: 1
MDPNVLRSDVFVEFLKLAQLVQF 69 Scare = 140 bits (301), Expect = 4e-31
Identities = 59/61 (96%) Frame = +1/+1 158P1H4: 496
VVSHKIGLCRELLGYFGLFLIRFGKEGKLSVVKKLADF- ELPYVSLGSSEVENCKVGLRKW 675
VVSHKIGLCRELLGYFGLFLIRFGKEGKLSVVKKLAD- FELPYVSLGSSEVENCKVGLRK
GenScan: 553 VVSHKIGLCRELLGYFGLFLIRFGKEGKLSV-
VKKLADFELPYVSLGSSEVENCKVGLRKC 732 Score = 248 bits (535), Expect =
2e-63 Identities = 100/105 (95%) Frame = +1/+1 158P1H4: 739
MQAIQDIEKGWAKPTQAQRQKLEAFQKEDSQTKFLELAREVRHYGYL- QLDPCTCDYPESG 918
+AIQDIEKGWAKPTQAQRQKLEAFQKEDSQTKFLELAREVRHYGYL- QLDPCTCDYPESG
GenScan: 892 LSAIQDIEKGWAKPTQAQRQKLEAFQKEDSQTKFLELARE-
VRHYGYLQLDPCTCDYPESG 1071 158P1H4: 919
SGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVTFLGTLL 1053
SGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVTFL ++L GenScan: 1072
SGAVLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVTFLVSIL 1206
[0886]
37TABLE XXIVB Peptide sequences from the translation of the
nucleotide sequence of the splice variant predicted Genscan. Open
reading frame Amino acid sequences Frame 1
MDPNVLRSDVFVEFLKLAQLVQFSTHLAALLKTSGSGLFS- FPPFASRLAAPAEGEDPALP
SFFRGSENCTEHRNLEDVPDRRRKGTQNMGERERQ- KEGEGNCFESGETIPQLLFIFKAML
RTPTHTSHLIRLLPGGNLCISDKRRPPVSLV- FVQLPPRELGVVGKAALEGSAWRPLLAWY
RGDLVVSHKIGLCRELLGYFGLFLIRF- GKEGKLSVVKKLADFELPYVSLGSSEVENCKVG
LRKCFGQVMEWSAGAAANIHTQV- TSCPRCSHSPHRHLRRPDLFQAWLHNSGKKAQKLLSA
IQDIEKGWAKPTQAQRQKLEAFQKEDSQTKFLELAREVRHYGYLQLDPCTCDYPESGSGA
VLSVGNNEISCCITLPDSQTQDIVFQMSRVKCWQVTFLVSILAPCFAHDLKILVPLDDKV
DTRETGLPFESVP* Frame 2 WTQTC*EVMSSLSF*NWRSWFNFQRT-
WQPF*KPQAQGYLASLHLLQD*QPQLKARTLPFL
PSSEGVRIALSIETWRTCQTGGGKEPKTWGKERDRKKGKGIALKVVKLFLSCSLFSKLC*
EHPLIPAT*SDCFLGVIYVSLTREGPL*AWYLYNCHLENWESWVKQLWRAVLGGLSLRGT
EVI*WCHTKLDCVESSWATSASFSFGLARRASSLL*KNWLTLNSLMLVLEVLRWKTVRLD
SESASAKSWNGQPVLPPTYTPRSPAAPGVHTHHTAIFGDLIFSRLGSTILGRKLRSFFQQ
YRTLKKDGPNPHRHRGRN*KLSRKKTVKQSFWSWPGRYGTMDTCSWILVPVTTQNQ- ALEL
FFLLAIMRSAAASPCLTARPRTSFSR*AG*SAGRSLSL*VSWHLALPMT*KS- *YPWMTKL
TLGRQVCHLNLYL Frame 3
GPKRVEK*CLR*VFKTGAAGSIFNALGSPSENLRLRAI*LPSICFKISSPS*RRGPCPSF
LLPRE*ELH*ASKPGGRARQEEERNPKHGGKRETERRGRELL*KW*NYSSALYFQSYAE
NTHSYQPPNQTASWG*FMYL*QEKAPCEPGICTTAT*RTGSRG*SSSGGQCLEASFCVVQ
R*FSGVTQNWTVSRALGLLRPLSHSVWQGGQALCCEKIG*L*TPLC*SWKF*GGKL*GWT
PKVLRPSHGMVSRCCRQHTHPGHQLPQVFTLTTPPSSET*SFPGLAPQFWEESSEA- SFSN
TGH*KRMGQTHTGTEAEIRSFPERRQSNKVFGAGPGGTALWIPAAGSLYL*L- PRIRLWSC
SFCWQ**DQLLHHPA*QPDPGHRFPDEQGEVLAGHFPCEYLGTLLCP*- LKNPSTLG*QS*
H*GDRFAI*ICTL Note: Frame 1 gives the longest subsequence that is
identical with 158P1H4 amino acid sequence. In this Table each (*)
indicates the product of a single codon, i.e., a single unknown
amino acid or a stop codon.
* * * * *
References